Antibodies directed to ERBB2

ABSTRACT

The present invention relates to antibodies including human antibodies and antigen-binding portions thereof that specifically bind to ErbB2, preferably human ErbB2. In another embodiment, the antibodies or antigen-binding portions thereof inhibit ErbB2. The invention also relates to antibodies that are chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulins or portions thereof derived from human anti-ErbB2 antibodies and nucleic acid molecules encoding such immunoglobulins. The present invention also relates to methods of using the antibodies and compositions for diagnosis and treatment. The invention also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-ErbB2 antibodies. The invention also relates to transgenic animals or plants comprising nucleic acid molecules of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/376,196 filed on Nov. 2, 2009, said application Ser. No. 12/376,196is a National Stage Application of PCT/US2007/075078 filed on Aug. 2,2007, said PCT/US2007/075078 claims priority under 35 U.S.C. §119(e) toU.S. Provisional Application No. 60/835,514 filed Aug. 4, 2006. Each ofthe above listed applications is incorporated by reference herein in itsentirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled ERBB2_Sequence_Listingcreated on Jun. 27, 2012 and having a size of 60.8 kilobytes.

FIELD OF THE INVENTION

The present invention concerns anti-ErbB2 antibodies particularly humanantibodies, and methods for making and using anti-ErbB2 antibodies, forexample to treat cancer.

BACKGROUND OF THE INVENTION

The ErbB family of receptor tyrosine kinases are important mediators ofcell growth, differentiation and survival. The receptor family includesfour distinct members including epidermal growth factor receptor (EGFRor ErbB1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4).

EGFR, encoded by the erbB1 gene, has been causally implicated in humanmalignancy. In particular, increased expression of EGFR has beenobserved in breast, bladder, lung, head, neck and stomach cancer as wellas glioblastomas. Increased EGFR receptor expression is often associatedwith increased production of the EGFR ligand, transforming growth factoralpha (TGF-α), by the same tumor cells resulting in receptor activationby an autocrine stimulatory pathway (Baselga and Mendelsohn, Pharmac.Ther. 64:127-154 (1994)). Monoclonal antibodies directed against theEGFR or its ligands, TGF-α and EGF, have been evaluated as therapeuticagents in the treatment of such malignancies. See, e.g., Baselga andMendelsohn, supra; Masui et al. Cancer Research 44:1002-1007 (1984); andWu et al. J. Clin. Invest. 95:1897-1905 (1995).

The second member of the ErbB family, p185neu, was originally identifiedas the product of the transforming gene from neuroblastomas ofchemically treated rats. The activated form of the neu proto-oncogeneresults from a point mutation (valine to glutamic acid) in thetransmembrane region of the encoded protein. Amplification of the humanhomolog of neu is observed in breast and ovarian cancers and correlateswith a poor prognosis (Slamon et al., Science, 235:177-182 (1987);Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No.4,968,603). To date, no point mutation analogous to that in the neuproto-oncogene has been reported for human tumors. Overexpression ofErbB2 (frequently but not uniformly due to gene amplification) has alsobeen observed in other carcinomas including carcinomas of the stomach,endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas andbladder. See, among others, King et al., Science, 229:974 (1985); Yokotaet al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol.,6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988); Cohen etal., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res.,51:1034:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990);Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res.,50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al.,Mol. Carcinog., 3:254-257 (1990); Aasland et al. Br. J. Cancer57:358-363 (1988); Williams et al. Pathobiology 59:46-52 (1991); andMcCann et al., Cancer, 65:88-92 (1990).

ErbB2 may be overexpressed in prostate cancer (Gu et al. Cancer Lett.99:185-9 (1996); Ross et al. Hum. Pathol. 28:827-33 (1997); Ross et al.Cancer 79:2162-70 (1997); and Sadasivan et al. J. Urol. 150:126-31(1993)).

Antibodies directed against the rat p185neu and human ErbB2 proteinproducts have been described. Drebin and colleagues have raisedantibodies against the rat neu gene product, p185neu. See, for example,Drebin et al., Cell 41:695-706 (1985); Myers et al., Meth. Enzym.198:277-290 (1991); and WO94/22478. Drebin et al. Oncogene 2:273-277(1988) report that mixtures of antibodies reactive with two distinctregions of p185neu result in synergistic anti-tumor effects onneu-transformed NIH-3T3 cells implanted into nude mice. See also U.S.Pat. No. 5,824,311 issued Oct. 20, 1998.

Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe thegeneration of a panel of anti-ErbB2 antibodies which were characterizedusing the human breast tumor cell line SK-BR-3. Relative cellproliferation of the SK-BR-3 cells following exposure to the antibodieswas determined by crystal violet staining of the monolayers after 72hours. Using this assay, maximum inhibition was obtained with theantibody called 4D5 which inhibited cellular proliferation by 56%. Otherantibodies in the panel reduced cellular proliferation to a lesserextent in this assay. The antibody 4D5 was further found to sensitizeErbB2-overexpressing breast tumor cell lines to the cytotoxic effects ofTNF-α. See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. Theanti-ErbB2 antibodies discussed in Hudziak et al. are furthercharacterized in Fendly et al. Cancer Research 50:1550-1558 (1990);Kotts et al. In vitro 26(3):59A (1990); Sarup et al. Growth Regulation1:72-82 (1991); Shepard et al. J. Clin. Immunol. 11(3):117-127 (1991);Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis et al. CancerImmunol. Immunother. 37:255-263 (1993); Pietras et al. Oncogene9:1829-1838 (1994); Vitetta et al. Cancer Research 54:5301-5309 (1994);Sliwkowski et al. J. Biol. Chem. 269(20):14661-14665 (1994); Scott etal. J. Biol. Chem. 266:14300-5 (1991); D'souza et al. Proc. Natl. Acad.Sci. 91:7202-7206 (1994); Lewis et al. Cancer Research 56:1457-1465(1996); and Schaefer et al. Oncogene 15:1385-1394 (1997).

A recombinant humanized version of the murine anti-ErbB2 antibody 4D5(huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®; U.S. Pat. No. 5,821,337) isclinically active in patients with ErbB2-overexpressing metastaticbreast cancers that have received extensive prior anti-cancer therapy(Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN® receivedmarketing approval from the Food and Drug Administration Sep. 25, 1998for the treatment of patients with metastatic breast cancer whose tumorsoverexpress the ErbB2 protein.

Other anti-ErbB2 antibodies with various properties have been describedin Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al.Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991);Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski etal. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993);WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancocket al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res.54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994);Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No.5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

Homology screening has resulted in the identification of two other ErbBreceptor family members; ErbB3 (U.S. Pat. Nos. 5,183,884 and 5,480,968as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989)) and ErbB4 (EPPat Appln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA,90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993)).Both of these receptors display increased expression on at least somebreast cancer cell lines.

The ErbB receptors are generally found in various combinations in cellsand heterodimerization is thought to increase the diversity of cellularresponses to a variety of ErbB ligands (Earp et al. Breast CancerResearch and Treatment 35: 115-132 (1995)). EGFR is bound by at leastsix different ligands; epidermal growth factor (EGF), transforminggrowth factor alpha (TGF-α), amphircgulin, heparin binding epidermalgrowth factor (HB-EGF), betacellulin and epiregulin (Groenen et al.Growth Factors 11:235-257 (1994)). A family of heregulin proteinsresulting from alternative splicing of a single gene are ligands forErbB3 and ErbB4. The heregulin family includes alpha, beta and gammaheregulins (Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat. No.5,641,869; and Schaefer et al. Oncogene 15:1385-1394 (1997)); neudifferentiation factors (NDFs), glial growth factors (GGFs);acetylcholine receptor inducing activity (ARIA); and sensory and motorneuron derived factor (SMDF). For a review, see Groenen et al. GrowthFactors 11:235-257 (1994); Lemke, G. Molec. & Cell. Neurosci. 7:247-262(1996) and Lee et al. Pharm. Rev. 47:51-85 (1995). Recently threeadditional ErbB ligands were identified; neuregulin-2 (NRG-2) which isreported to bind either ErbB3 or ErbB4 (Chang et al. Nature 387 509-512(1997); and Carraway et al Nature 387:512-516 (1997)); neuregulin-3which binds ErbB4 (Zhang et al. PNAS (USA) 94(18):9562-7 (1997)); andneuregulin-4 which binds ErbB4 (Harari et al. Oncogene 18:2681-89(1999)) HB-EGF, betacellulin and epiregulin also bind to ErbB4. WhileEGF and TGFα do not bind ErbB2, EGF stimulates EGFR and ErbB2 to form aheterodimer, which activates EGFR and results in transphosphorylation ofErbB2 in the heterodimer. Dimerization and/or transphosphorylationappear to activate the ErbB2 tyrosine kinase. See Earp et al., supra.Likewise, when ErbB3 is co-expressed with ErbB2, an active signalingcomplex is formed and antibodies directed against ErbB2 are capable ofdisrupting this complex (Sliwkowski et al., J. Biol. Chem.,269(20):14661-14665 (1994)). Additionally, the affinity of ErbB3 forheregulin (HRG) is increased to a higher affinity state whenco-expressed with ErbB2. See also, Levi et al., Journal of Neuroscience15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92:1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996)with respect to the ErbB2-ErbB3 protein complex. ErbB4, like ErbB3,forms an active signaling complex with ErbB2 (Carraway and Cantley, Cell78:5-8 (1994)).

The product of the HER-2/neu proto-oncogene, HER2, is the second memberof the human epidermal growth factor receptor (HER) family of tyrosinekinase receptors and has been suggested to be a ligand orphan receptor.Ligand-dependent heterodimerization between HER2 and another HER familymember, HER1, HER3 or HER4, activates the HER2 signaling pathway. Theintracellular signaling pathway of HER2 is thought to involve ras-MAPKand PI3K pathways, as well as MAPK-independent S6 kinase andphospholipase C-gamma signaling pathways (Graus-Porta et al., Mol CellBiol. 1995 March; 15(3): 1182-1191; Grant et al., Front Biosci. 2002Feb. 1; 7:d376-89).

HER2 signaling also effects proangiogenic factors, vascular endothelialgrowth factor (VEGF) and interleukin-8 (IL-8), and an antiangiogenicfactor, thrombospondin-1 (TSP-1). Re-expression of HER2 in MCF-7 andT-47D breast cancer cells that endogenously express low levels of HER2results in elevated expression of VEGF and IL-8 and decreased expressionof TSP-1. Inhibition of HER2 with a humanized anti-HER2 antibody(trastuzumab, or Herceptin®) or a retrovirus-mediated small interferingRNA against HER2 (siHER2) decreases VEGF and IL-8 expression, butincreased TSP-1 expression in BT474 breast cancer cells that expresshigh levels of HER2. HER2 signaling therefore influences the equilibriumbetween pro- and antiangiogenic factors via distinct signaling pathways.Trastuzumab inhibits angiogenesis and tumor growth, at least in part,through activation of the HER2-p38-TSP-1 pathway and inhibition of theHER2—PI3K-AKT-VEGF/IL-8 pathway (Wen et al., Oncogene. 2006 May 22;Epub). Additionally, ErbB2 membrane RTK can confer resistance totaxol-induced apoptosis by directly phosphorylating Cdc2 (Tan et al.,Mol. Cell. 2002 May; 9 (5):993-1004).

SUMMARY OF THE INVENTION

Certain embodiments of the invention are described below.

In one embodiment, the invention comprises a targeted binding agent,e.g. a human monoclonal antibody or an antigen-binding portion thereof,that specifically binds to ErbB2. The targeted binding agent can possessat least one of the following properties:

-   -   (a) binds to human cells;    -   (b) binds to cells expressing cynomolgus ErbB2;    -   (c) competes partially with Herceptin® but does not compete with        2C4;    -   (d) inhibits ErbB2 phosphorylation in MCF7 cells with a EC50 of        less than 50 ng/ml;    -   (e) inhibits cell proliferation with an EC50 of less than 50        ng/ml in SKBR3 cells;    -   (f) binds to ErbB2 with a KD of 13.5 nM or less; or    -   (g) has an off rate (koff) for ErbB2 of 2.14×10-4 s-1 or        smaller.        In another embodiment, the targeted binding agent binds ErbB2        with a KD of 13.5 nM or less and inhibits activation of ErbB. In        another embodiment, said targeted binding agent is an antibody.        In an embodiment, said antibody is a humanized, chimeric or        human monoclonal antibody or antigen-binding portion thereof.

In another embodiment, the invention comprises a targeted binding agent,e.g. a humanized, chimeric or human monoclonal antibody orantigen-binding portion thereof, that binds specifically to and inhibitshuman ErbB2, wherein the targeted binding agent has at least oneproperty selected from the group consisting of:

(a) competes for binding to ErbB2 with an antibody selected from thegroup consisting of: 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.3;

(b) competes for binding to ErbB2 with an antibody selected from thegroup consisting of: 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.3;

(c) binds to the same epitope of ErbB2 as an antibody selected from thegroup consisting of: 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.3;

(d) binds to ErbB2 with substantially the same KD as an antibodyselected from the group consisting of: 1.44.1, 1.140, 1.43, 1.14.1,1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43,1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3; and

(e) binds to ErbB2 with substantially the same off rate as an antibodyselected from the group consisting of: 1.44.1, 1.140, 1.43, 1.14.1,1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43,1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3.

In another embodiment, said binding targeted agent is an antibody. Inanother embodiment, said antibody is a humanized, chimeric or humanmonoclonal antibody or antigen-binding portion thereof.

In another embodiment, the invention comprises a targeted binding agent,e.g. a monoclonal antibody or an antigen-binding portion thereof, thatspecifically binds ErbB2, wherein:

-   -   (a) the targeted binding agent comprises the heavy chain CDR1,        CDR2 and CDR3 amino acid sequences of an antibody selected from        the group consisting of: 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1,        1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43,        1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3;    -   (b) the targeted binding agent comprises the light chain CDR1,        CDR2 and CDR3 amino acid sequences of an antibody selected from        the group consisting of 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1,        1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43,        1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3; and    -   (c) the targeted binding agent comprises a heavy chain of (a)        and a light chain of (b); or the targeted binding agent of (c)        wherein the heavy chain and light chain CDR amino acid sequences        are selected from the same antibody.        In another embodiment, said targeted binding agent is an        antibody. In another embodiment, said antibody is a humanized,        chimeric or human monoclonal antibody or antigen-binding portion        thereof. In another embodiment, the monoclonal antibody or an        antigen-binding portion thereof further includes the heavy chain        amino acid sequence set forth in SEQ ID NO: 46, the light chain        amino acid sequence set forth in SEQ ID NO: 47, or both.

In another embodiment, the monoclonal antibody or an antigen-bindingportion thereof of the invention can be from any isotype.

In another embodiment, the human monoclonal antibody or antigen-bindingportion thereof of the invention includes a heavy chain that utilizes ahuman VH 3-21 gene, a human VH 3-7 gene, a human VH 4-31 gene, or ahuman VH 3-13 gene.

In another embodiment, the human monoclonal antibody or antigen-bindingportion thereof of the invention includes a heavy chain that utilizes ahuman VH 3-21 gene, a human VH 3-7 gene, a human VH 4-31 gene, or ahuman VH 3-13 gene.

In another embodiment, the human monoclonal antibody or antigen-bindingportion thereof of the invention includes a light chain that utilizes ahuman VK B3 gene, a human VK L1 gene, a human VK A2 gene, or a human VKA1 gene.

In another embodiment, the human monoclonal antibody or antigen-bindingportion thereof of the invention includes an antibody or portion furthercomprises a light chain that utilizes a human VK B3 gene, a human VK L1gene, a human VK A2 gene, or a human VK A1 gene.

In another embodiment, the VL domain, the VH domain, or both of thehuman monoclonal antibody or antigen-binding portion thereof of theinvention are at least 90% identical in amino acid sequence to the VLdomain, VH domain or both, respectively, of monoclonal antibodies1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39,1.24 and 1.71.3

In another embodiment, the invention includes a monoclonal antibody oran antigen-binding portion thereof that specifically binds ErbB2,wherein the antibody comprises one or more of an FR1, FR2, FR3 or FR4amino acid sequence of an antibody selected from the group consistingof: 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20,1.39, 1.24 and 1.71.3.

In another embodiment, the invention includes a human monoclonalantibody or an antigen-binding portion, wherein the antibody includes

-   -   (a) a heavy chain amino acid sequence that is at least 90%        identical to the heavy chain amino acid sequence of monoclonal        antibody 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,        1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1,        1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3;    -   (b) a light chain amino acid sequence that is at least 90%        identical to the light chain amino acid sequence of monoclonal        antibody 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,        1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1,        1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3; or both (a) and (b).

In another embodiment, the invention includes a human monoclonalantibody or an antigen-binding portion described herein, wherein saidantigen-binding portion is selected from the group consisting of: Fab,Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity determining region (CDR)fragments, single-chain antibodies (scFv or scFv2), chimeric antibodies,diabodies, dispecific antibodies, and polypeptides that contain at leasta portion of an antibody that is sufficient to confer specific antigenbinding to the polypeptide

In yet another embodiment, the invention includes a compositioncomprising the targeted binding agent, e.g. an antibody orantigen-binding portion described herein, and a pharmaceuticallyacceptable carrier. The composition can further include an additionaltherapeutic or diagnostic agent. In one embodiment, the compositionfurther includes a second antibody that specifically binds ErbB2 whereinsaid second antibody does not compete for binding to ErbB2 with anantibody selected from the group consisting of: 1.14.1, 1.18.1, 1.20.1,1.24.3, 1.39.1, 1.71.3, 1.96.2, 1.100.1, and 1.140.1.

In yet another embodiment, the invention includes an isolated cell linethat produces the targeted binding agent, antibody or antigen-bindingportion described herein or the heavy chain or light chain of saidantibody or said portion.

In yet another embodiment, the invention includes an isolated nucleicacid molecule comprising a nucleotide sequence that encodes the heavychain or an antigen-binding portion thereof, the light chain or anantigen-binding portion thereof, or both. The isolated nucleic acid caninclude a nucleotide sequence selected from the group consisting of:

-   -   (a) the nucleotide sequence of SEQ ID NO:1;    -   (b) the nucleotide sequence encoding SEQ ID NO:2;    -   (c) the nucleotide sequence of SEQ ID NO:3;    -   (d) the nucleotide sequence encoding SEQ ID NO:4;    -   (e) the nucleotide sequence of SEQ ID NO:5;    -   (f) the nucleotide sequence encoding SEQ ID NO:6;    -   (g) the nucleotide sequence of SEQ ID NO:7;    -   (h) the nucleotide sequence encoding SEQ ID NO:8;    -   (i) the nucleotide sequence of SEQ ID NO:9;    -   (j) the nucleotide sequence encoding SEQ ID NO:10;    -   (k) the nucleotide sequence of SEQ ID NO:11;    -   (l) the nucleotide sequence encoding SEQ ID NO:12;    -   (m) the nucleotide sequence of SEQ ID NO:13;    -   (n) the nucleotide sequence encoding SEQ ID NO:14;    -   (o) the nucleotide sequence of SEQ ID NO:15;    -   (p) the nucleotide sequence encoding SEQ ID NO:16;    -   (q) the nucleotide sequence of SEQ ID NO:17;    -   (r) the nucleotide sequence encoding SEQ ID NO:18;    -   (s) the nucleotide sequence of SEQ ID NO:19;    -   (t) the nucleotide sequence encoding SEQ ID NO:20;    -   (u) the nucleotide sequence of SEQ ID NO:21;    -   (v) the nucleotide sequence encoding SEQ ID NO:22;    -   (w) the nucleotide sequence of SEQ ID NO:23;    -   (x) the nucleotide sequence encoding SEQ ID NO:24;    -   (y) the nucleotide sequence of SEQ ID NO:25;    -   (z) the nucleotide sequence encoding SEQ ID NO:26;    -   (aa) the nucleotide sequence of SEQ ID NO:27;    -   (bb) the nucleotide sequence encoding SEQ ID NO:28;    -   (cc) the nucleotide sequence of SEQ ID NO:29;    -   (dd) the nucleotide sequence encoding SEQ ID NO:30;    -   (ee) the nucleotide sequence of SEQ ID NO:31;    -   (ff) the nucleotide sequence encoding SEQ ID NO:32;    -   (gg) the nucleotide sequence of SEQ ID NO:33;    -   (hh) the nucleotide sequence encoding SEQ ID NO:34;    -   (ii) the nucleotide sequence of SEQ ID NO:35;    -   (jj) the nucleotide sequence encoding SEQ ID NO:36;    -   (kk) the nucleotide sequence of SEQ ID NO:37;    -   (ll) the nucleotide sequence encoding SEQ ID NO:38;    -   (mm) the nucleotide sequence of SEQ ID NO:39;    -   (nn) the nucleotide sequence encoding SEQ ID NO:40;    -   (oo) the nucleotide sequence of SEQ ID NO:41;    -   (pp) the nucleotide sequence encoding SEQ ID NO:42;    -   (qq) the nucleotide sequence of SEQ ID NO:43; and    -   (rr) the nucleotide sequence encoding SEQ ID NO:44.

The invention further comprises a vector comprising the nucleic acidmolecule described herein, wherein the vector optionally comprises anexpression control sequence operably linked to the nucleic acidmolecule. In another embodiment, the invention includes a host cellcomprising the vector or the nucleic acid molecule described herein.

In another embodiment, the invention includes a method for producing thetargeted bindging agent, monoclonal antibody or antigen-binding portiondescribed herein comprising the steps of culturing the host cell or thecell line described herein under suitable conditions and recovering saidantibody or antigen-binding portion.

The invention further comprises a non-human transgenic animal ortransgenic plant comprising the nucleic acid described herein, whereinthe non-human transgenic animal or transgenic plant expresses saidnucleic acid. The invention also includes a method for isolating anantibody or antigen-binding portion thereof that specifically binds tohuman ErbB2. The method includes the step of isolating the antibody fromthe non-human transgenic animal or transgenic plant.

In another embodiment, the invention includes a method for making ahuman monoclonal antibody that specifically binds to ErbB2, comprisingthe steps of:

-   -   (i) immunizing a non-human transgenic animal that is capable of        producing human antibodies with ErbB2, an immunogenic portion of        ErbB2 or a cell or tissue expressing ErbB2;    -   (ii) allowing the transgenic animal to mount an immune response        to ErbB2; and    -   (iii) recovering the antibody.

In another embodiment, the invention includes a method for treating,preventing or alleviating the symptoms of an ErbB2-mediated disorder ina subject in need thereof, comprising the step of administering to saidsubject a targeted binding agent, an antibody or antigen-binding portionor the composition described herein, wherein said targeted bindingagent, antibody or antigen-binding portion inhibit ErbB2.

In yet another aspect, the invention includes a method for treating,preventing or alleviating the symptoms of an ErbB2-mediated disordersuch as cancer in a subject in need thereof with a targeted bindingagent, e.g. an antibody or antigen-binding portion thereof, thatspecifically binds to ErbB2 comprising the steps of:

-   -   (i) administering an effective amount of an isolated nucleic        acid molecule encoding the heavy chain or the antigen-binding        portion thereof, an isolated nucleic acid molecule encoding the        targeted bind agent, e.g. the light chain or the antigen-binding        portion thereof, or both the nucleic acid molecules encoding the        light chain and the heavy chain or antigen-binding portions        thereof of an antibody; and,    -   (ii) expressing the nucleic acid molecule.

An ErbB2-mediated disorder can be selected from the group consisting ofbreast, bladder, lung, head, neck, prostate, stomach, endometrium,salivary gland, lung, kidney, colon, thyroid, pancreatic cancer, andglioblastomas

In another embodiment, the invention includes a method of inhibitingproliferation of a cancer cell expressing ErbB2 in a subject in needthereof, the method comprising the step of administering to said subjecta targeting binding agent, e.g. an antibody or antigen-binding portionor the composition described herein, wherein said targeted binding agentinhibits ErbB2. In another embodiment, said binding targeted agent is anantibody. In another embodiment, said antibody is a humanized, chimericor human monoclonal antibody or antigen-binding portion thereof.

In another embodiment, the invention includes a method for inhibiting anErbB2 activity in a cell, for example, of a subject or cancer cell,expressing ErbB2, comprising contacting the cell with a targeted bindingagent or with the composition described herein, wherein the ErbB2activity in the cell is selected from the group consisting of:

-   -   (a) phosphorylation of ErbB2;    -   (b) activation of the MAPK pathway;    -   (c) activation of the PI3K pathway;    -   (d) inhibition of CDC2; and    -   (e) combinations thereof.        In one embodiment, the phosphorylation of ErbB2 is inhibited by        48 hours. In another embodiment, said binding targeted agent is        an antibody. In another embodiment, said antibody is a        humanized, chimeric or human monoclonal antibody or        antigen-binding portion thereof.

In another embodiment, the invention includes a method for modulating anErbB2 activity in a cell expressing ErbB2, including contacting the cellwith an antibody or antigen-binding portion or with the compositiondescribed herein wherein the ErbB2 activity in the cell is activation ofthe p38-TSP-1 pathway.

In one embodiment an antigen binding site may comprise a heavy chainCDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3 of any ofantibodies 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20,1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,1.20, 1.39, 1.24 and 1.71.3 with as many as twenty, sixteen, ten, nineor fewer, e.g. one, two, three, four or five, amino acid additions,substitutions, deletions, and/or insertions within the disclosed CDRs.Such modifications may potentially be made at any residue within theCDRs.

In another embodiment the targeted binding agent or antibody maycomprise a sequence comprising any one, two, three, four, five or six ofthe CDR1, CDR2 or CDR3 sequences as shown in Table 4, 4(a) and/or Table5. In another embodiment the targeted binding agent or antibody maycomprise a sequence comprising a CDR1, CDR2 and CDR3 sequence as shownin Table 4, and/or 4(a). In another embodiment the targeted bindingagent or antibody may comprise a sequence comprising a CDR1, CDR2 andCDR3 sequence as shown in Table 5. In another embodiment the targetedbinding agent or antibody may comprise a sequence comprising a CDR1,CDR2 and CDR3 sequence as shown in Table 4 or 3(a) and a CDR1, CDR2 andCDR3 sequence as shown in Table 5. It is noted that those of ordinaryskill in the art can readily accomplish CDR determinations. See forexample, Kabat et al., Sequences of Proteins of Immunological Interest,Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3,or as defined herein.

In another embodiment the targeted binding agent or antibody maycomprise a sequence comprising any one, two, three, four, five or six ofthe CDR1, CDR2 and CDR3 sequences of any one of the fully humanmonoclonal antibodies 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3, as shown in Table 4, 4(a) orin Table 5. In one embodiment the targeted binding agent or antibody maycomprise a sequence comprising a CDR1, CDR2 and CDR3 sequence of fullyhuman monoclonal antibody 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3 as shown in Table 4 and 4(a).In another embodiment the targeted binding agent or antibody maycomprise a sequence comprising a CDR1, CDR2 and CDR3 sequence of fullyhuman monoclonal antibody 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3, as shown in Table 5. Inanother embodiment the targeted binding agent or antibody may comprise asequence comprising a CDR1, CDR2 and CDR3 sequence of fully humanmonoclonal antibody 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.3 as shown in Table 4 and 4(a), and aCDR1, CDR2 and CDR3 sequence of fully human monoclonal antibody 1.44.1,1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39,1.24 and 1.71.3 as shown in Table 5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the correlation of the effects of 152hybridoma supernatants on Heregulin-induced ErbB2 phosphorylation inMCF7 cells after 1 hr (Y axis) and 24 hr (X axis) pre-incubation.

FIG. 2 is a data plot of LA/HA ELISA analysis. The Y axis indicates thebinding signal in OD value when 31 ng/ml of hErbB2(ECD)/cMyc-His wascoated on ELISA plate. The X axis indicates the concentration ofErbB2-specific antibodies in hybridoma supernatants derived from HAELISA when 10 μg/ml of ErbB2(ECD)/cMyc-His was coated on the ELISAplate.

FIGS. 3A-3C show dose response curves for 10 anti-ErbB2 monoclonalantibodies of the invention (B and C) and control antibodies (A) inHeregulin-induced ErbB2 phosphorylation in MCF7 cells.

FIGS. 4A-4C show dose response curves for 10 anti-ErbB2 monoclonalantibodies of the invention (B and C) and control antibodies (A) inHeregulin-induced proliferation of MCF7 cells.

FIGS. 5A and 5B show dose response curves for 8 anti-ErbB2 monoclonalantibodies of the invention (B) and control antibodies (A) in a BT474cell proliferation assay.

FIGS. 6A and 6B show dose response curves for 8 anti-ErbB2 monoclonalantibodies of the invention (B) and control antibodies (A) in an SKBR3cell proliferation assay.

FIGS. 7A-7C show dose-dependent response of total ErbB2 phosphorylationto mAb 1.18.1 (A), Herceptin® (B) and 2C4 (C) after incubating cellswith mAbs for 24, 48, 72, or 96 hours.

FIGS. 8A-8C show dose-dependent response of normalized ErbB2phosphorylation to mAb 1.18.1 (A), Herceptin® (B) and 2C4 (C) afterincubating cells with mAbs for 24, 48, 72, or 96 hours

FIGS. 9A and 9B show the results of competition binning indicating thatthe tested antibodies of the invention do not compete with 2C4 (B) orHerceptin® (A) for ErbB2 binding in an ELISA.

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofsubjects.

Each antibody has been given an identification number that includeseither two or three numbers separated by one or two decimal points. Insome cases, several clones of one antibody were prepared. A number ofclones have different identification numbers although they have theidentical nucleic acid and amino acid sequences as the parent sequence,they may also be listed separately. Thus, for example, the nucleic acidand amino acid sequences for:

1.44.1=1.44.2=1.44.3=1.44;

1.124=1.148=1.140=1.140.1;

1.41=1.43=143.1=143.2=1.22.1;

1.14.1=1.14.2=1.14.3=1.14;

1.100.1=1.100.2=1.100.3=1.100;

1.107=1.104=1.128=1.96=1.99=1.96.2;

1.18.1=1.18.2=1.18.3=1.18;

1.20=1.19=1.20.1;

1.39=1.39.1=1.39.2=1.39.3;

1.24=1.22.2=1.71.1=1.24.3;

1.71.2=1.71.3;

are identical.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein”, “isolated polypeptide” or “isolatedantibody” is a protein, polypeptide or antibody that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) is freeof other proteins from the same species, (3) is expressed by a cell froma different species, or (4) does not occur in nature. Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A protein mayalso be rendered substantially free of naturally associated componentsby isolation, using protein purification techniques well known in theart.

Examples of isolated antibodies include an anti-ErbB2 antibody that hasbeen affinity purified using ErbB2, an anti-ErbB2 antibody that has beensynthesized by a hybridoma or other cell line in vitro, and a humananti-ErbB2 antibody derived from a transgenic mouse. Targeted bindingagents can also be purified using similar techniques described herein.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence. In some embodiments,fragments are at least 5, 6, 8 or 10 amino acids long. In otherembodiments, the fragments are at least 14, at least 20, at least 50, orat least 70, 80, 90, 100, 150 or 200 amino acids long.

The term “modulating” as used herein refers to any amount of inhibitionor activation of a pathway.

In certain embodiments, amino acid substitutions to an anti-ErbB2antibody or antigen-binding portion thereof are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, and (4) conferor modify other physicochemical or functional properties of suchanalogs, but still retain specific binding to ErbB2. Analogs can includevarious muteins of a sequence other than the normally-occurring peptidesequence. For example, single or multiple amino acid substitutions,preferably conservative amino acid substitutions, may be made in thenormally-occurring sequence, preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence; e.g., a replacementamino acid should not alter the anti-parallel β-sheet that makes up theimmunoglobulin binding domain that occurs in the parent sequence, ordisrupt other types of secondary structure that characterizes the parentsequence. In general, glycine and proline would not be used in ananti-parallel β-sheet. Examples of art-recognized polypeptide secondaryand tertiary structures are described in Proteins, Structures andMolecular Principles (Creighton, Ed., W.H. Freeman and Company, New York(1984)); Introduction to Protein Structure (C. Branden and J. Tooze,eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al.,Nature 354:105 (1991), incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber andFreidinger, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229(1987), incorporated herein by reference. Such compounds are oftendeveloped with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a desiredbiochemical property or pharmacological activity), such as a humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH2NH—, —CH2S—,—CH═CH—(cis and trans), —COCH2—, —CH(OH)CH2—, and —CH2SO—, by methodswell known in the art. Systematic substitution of one or more aminoacids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

Where an “antibody” is referred to herein with respect to the invention,it is normally understood that an antigen-binding portion thereof mayalso be used. An antigen-binding portion competes with the intactantibody for specific binding. See generally, Fundamental Immunology,Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated byreference in its entirety for all purposes). Antigen-binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. In some embodiments,antigen-binding portions include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, andcomplementarity determining region (CDR) fragments, single-chainantibodies (scFv or scFv2), chimeric antibodies, diabodies andpolypeptides that contain at least a portion of an antibody that issufficient to confer specific antigen binding to the polypeptide.

From N-terminus to C-terminus, both the mature light and heavy chainvariable domains comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain herein is inaccordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) orChothia et al., Nature 342:878-883 (1989).

As used herein, an antibody that is referred to by number is the same asa monoclonal antibody that is obtained from the hybridoma of the samenumber. For example, monoclonal antibody 1.18 is the same antibody asone obtained from hybridoma 1*18, or a subclone thereof. Subclones areidentified with a further decimal, for example 1.18.1.

As used herein, an Fd fragment means an antibody fragment that consistsof the VH and CH1 domains; an Fv fragment consists of the VL and VHdomains of a single arm of an antibody; and a dAb fragment (Ward et al.,Nature 341:544-546 (1989)) consists of a VH domain.

In some embodiments, the antibody is a single-chain antibody (scFv) inwhich a VL and VH domains are paired to form a monovalent molecules viaa synthetic linker that enables them to be made as a single proteinchain. (Bird et al., Science 242:423-426 (1988) and Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988).) In some embodiments, theantibodies are diabodies, i.e., are bivalent antibodies in which VH andVL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites. (Seee.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448(1993), and Poljak R. J. et al., Structure 2:1121-1123 (1994).) In someembodiments, one or more CDRs from an antibody of the invention may beincorporated into a molecule either covalently or noncovalently to makeit an immunoadhesin that specifically binds to ErbB2. In suchembodiments, the CDR(s) may be incorporated as part of a largerpolypeptide chain, may be covalently linked to another polypeptidechain, or may be incorporated noncovalently.

In embodiments having one or more binding sites, the binding sites maybe identical to one another or may be different.

As used herein, the term “human antibody” means any antibody in whichthe variable and constant domain sequences are human sequences. The termencompasses antibodies with sequences derived from human genes, butwhich have been changed, e.g. to decrease possible immunogenicity,increase affinity, eliminate cysteines that might cause undesirablefolding, etc. The term encompasses such antibodies producedrecombinantly in non-human cells, which might impart glycosylation nottypical of human cells. These antibodies may be prepared in a variety ofways, as described below.

The term “chimeric antibody” as used herein means an antibody thatcomprises regions from two or more different antibodies. In oneembodiment, one or more of the CDRs of the chimeric antibody are derivedfrom a human anti-ErbB2 antibody. In another embodiment, all of the CDRsare derived from a human anti-ErbB2 antibody. In another embodiment, theCDRs from more than one human anti-ErbB2 antibodies are combined in achimeric antibody. For instance, a chimeric antibody may comprise a CDR1from the light chain of a first human anti-ErbB2 antibody, a CDR2 fromthe light chain of a second human anti-ErbB2 antibody and a CDR3 fromthe light chain of a third human anti-ErbB2 antibody, and CDRs from theheavy chain may be derived from one or more other anti-ErbB2 antibodies.Further, the framework regions may be derived from one of the anti-ErbB2antibodies from which one or more of the CDRs are taken or from one ormore different human antibodies.

In some embodiments, a chimeric antibody of the invention is a humanizedanti-ErbB2 antibody. A humanized anti-ErbB2 antibody of the inventioncomprises the amino acid sequence of one or more framework regionsand/or the amino acid sequence from at least a portion of the constantregion of one or more human anti-ErbB2 antibodies of the invention andCDRs derived from a non-human anti-ErbB2 antibody.

An “inhibiting antibody” (also referred to herein as an “antagonistantibody”) as used herein means an antibody that inhibits one or moreErbB2 activities by at least about 30% when added to a cell, tissue ororganism expressing ErbB2. In some embodiments, the antibody inhibitsErbB2 activity by at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%or greater than 100%. In some embodiments, the inhibiting antibody isadded in the presence of ligand such as heregulin. In some embodiments,an antagonist antibody of the invention decreases at least one activityof ErbB2 by 5-fold.

“Binding fragments” of an antibody are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)7, Fv, dAb and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a receptor to acounter-receptor when an excess of antibody reduces the quantity ofreceptor bound to counter-receptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

Fragments or analogs of antibodies or immunoglobulin molecules can bereadily prepared by those of ordinary skill in the art following theteachings of this specification. Preferred amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. Preferably, computerized comparison methods are usedto identify sequence motifs or predicted protein conformation domainsthat occur in other proteins of known structure and/or function. Methodsto identify protein sequences that fold into a known three-dimensionalstructure are known. See Bowie et al., Science 253:164 (1991).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (Ward,E. S. et al., (1989) Nature 341, 544-546) the Fab fragment consisting ofVL, VH, CL and CH1 domains; (McCafferty et al (1990) Nature, 348,552-554) the Fd fragment consisting of the VH and CH1 domains; (Holt etal (2003) Trends in Biotechnology 21, 484-490) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989), McCafferty etal (1990) Nature, 348, 552-554, Holt et al (2003) Trends inBiotechnology 21, 484-490], which consists of a VH or a VL domain; (v)isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site (Bird et al, (1988) Science, 242, 423-426, et al, (1988)PNAS USA, 85, 5879-5883); (viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; Holliger, P. (1993) etal, Proc. Natl. Acad. Sci. USA 90 6444-6448,). Fv, scFv or diabodymolecules may be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter, Y. et al, Nature Biotech, 14,1239-1245, 1996). Minibodies comprising a scFv joined to a CH3 domainmay also be made (Hu, S. et al, (1996) Cancer Res., 56, 3055-3061).Other examples of binding fragments are Fab′, which differs from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain, including one or more cysteines from theantibody hinge region, and Fab′-SH, which is a Fab′ fragment in whichthe cysteine residue(s) of the constant domains bear a free thiol group.

An “inhibiting targeted binding agent” as used herein means a targetedbinding agent that inhibits one or more ErbB2 activities by at leastabout 30% when added to a cell, tissue or organism expressing ErbB2. Insome embodiments, the targeted binding agent inhibits ErbB2 activity byat least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or greaterthan 100%. In some embodiments, the inhibiting targeted binding agent isadded in the presence of ligand such as heregulin. In some embodiments,the targeted binding agent of the invention decreases at least oneactivity of ErbB2 by 5-fold.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson U. et al., Ann. Biol. Clin. 51:19-26(1993); Jonsson U. et al., Biotechniques 11:620-627 (1991); Jonsson B.et al., J. Mol. Recognit. 8:125-131 (1995); and Johnsson B. et al.,Anal. Biochem. 198:268-277 (1991).

The term “KD” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor or otherwise interactingwith a molecule. Epitopic determinants generally consist of chemicallyactive surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be “linear” or “conformational.” In alinear epitope, all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearly along theprimary amino acid sequence of the protein. In a conformational epitope,the points of interaction occur across amino acid residues on theprotein that are separated from one another. An antibody is said tospecifically bind an antigen when the dissociation constant is ≦1 mM,preferably ≦100 nM and most preferably ≦10 nM. In certain embodiments,the KD is 1 pM to 500 pM. In other embodiments, the KD is between 500 pMto 1 μM. In other embodiments, the KD is between 1 μM to 100 nM. Inother embodiments, the KD is between 100 mM to 10 nM. Once a desiredepitope on an antigen is determined, it is possible to generateantibodies to that epitope, e.g., using the techniques described in thepresent invention. Alternatively, during the discovery process, thegeneration and characterization of antibodies may elucidate informationabout desirable epitopes. From this information, it is then possible tocompetitively screen antibodies for binding to the same epitope. Anapproach to achieve this is to conduct cross-competition studies to findantibodies that competitively bind with one another, e.g., theantibodies compete for binding to the antigen. A high throughput processfor “binning” antibodies based upon their cross-competition is describedin International Patent Application No. WO 03/48731.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Oren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), incorporated herein by reference.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms.

The term “isolated polynucleotide” as used herein means a polynucleotideof genomic, cDNA, or synthetic origin or some combination thereof, whichby virtue of its origin the “isolated polynucleotide” (1) is notassociated with all or a portion of a polynucleotides with which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “naturally occurring nucleotides” as used herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” as used herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. AcidsRes. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984);Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-CancerDrug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann andPeyman, Chemical Reviews 90:543 (1990), the disclosures of which arehereby incorporated by reference. An oligonucleotide can include a labelfor detection, if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest. The term “expression control sequence” as used herein meanspolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Insome embodiments, the vector is a plasmid, i.e., a circular doublestranded piece of DNA into which additional DNA segments may be ligated.In some embodiments, the vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. In some embodiments,the vectors are capable of autonomous replication in a host cell intowhich they are introduced (e.g., bacterial vectors having a bacterialorigin of replication and episomal mammalian vectors). In otherembodiments, the vectors (e.g., non-episomal mammalian vectors) can beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”).

The term “recombinant host cell” (or simply “host cell”), as usedherein, means a cell into which a recombinant expression vector has beenintroduced. It should be understood that “recombinant host cell” and“host cell” mean not only the particular subject cell but also theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. “High stringency” or “highly stringent” conditions can beused to achieve selective hybridization conditions as known in the artand discussed herein. One example of “high stringency” or “highlystringent” conditions is the incubation of a polynucleotide with anotherpolynucleotide, wherein one polynucleotide may be affixed to a solidsurface such as a membrane, in a hybridization buffer of 6×SSPE or SSC,50% formamide, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA at a hybridization temperature of 42° C. for12-16 hours, followed by twice washing at 55° C. using a wash buffer of1×SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.

The term “CDR region” or “CDR” is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulin as definedby Kabat et al. 1991 (Kabat, E. A. et al. (1991) Sequences of Proteinsof Immunological Interest, 5th Edition. US Department of Health andHuman Services, Public Service, NIH, Washington), and later editions, oras defined herein. An antibody typically contains 3 heavy chain CDRs and3 light chain CDRs. The term CDR or CDRs is used here in order toindicate, according to the case, one of these regions or several, oreven the whole, of these regions which contain the majority of the aminoacid residues responsible for the binding by affinity of the antibodyfor the antigen or the epitope which it recognizes.

Among the six short CDR sequences, the third CDR of the heavy chain(HCDR3) has a greater size variability (greater diversity essentiallydue to the mechanisms of arrangement of the genes which give rise toit). It may be as short as 2 amino acids although the longest size knownis 26. CDR length may also vary according to the length that can beaccommodated by the particular underlying framework. Functionally, HCDR3plays a role in part in the determination of the specificity of theantibody (Segal et al., PNAS, 71:4298-4302, 1974, Amit et al., Science,233:747-753, 1986, Chothia et al., J. Mol. Biol., 196:901-917, 1987,Chothia et al., Nature, 342:877-883, 1989, Caton et al., J. Immunol.,144:1965-1968, 1990, Sharon et al., PNAS, 87:4814-4817, 1990, Sharon etal., J. Immunol., 144:4863-4869, 1990, Kabat et al., J. Immunol.,147:1709-1719, 1991).

The term a “set of CDRs” referred to herein comprises CDR1, CDR2 andCDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a setof LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a“set of CDRs” includes HCDRs and LCDRs.

The term “percent sequence identity” in the context of nucleotidesequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides,usually at least about 18 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or Bestfit,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996);Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein byreference). Unless otherwise specified, default parameters for aparticular program or algorithm are used. For instance, percent sequenceidentity between nucleotide sequences can be determined using FASTA withits default parameters (a word size of 6 and the NOPAM factor for thescoring matrix) or using Gap with its default parameters as provided inGCG Version 6.1, incorporated herein by reference.

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence.

As used herein, the terms “percent sequence identity” and “percentsequence homology” are used interchangeably.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, means that whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 85%, preferably at leastabout 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99%of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights as supplied with the programs,share at least 70%, 75% or 80% sequence identity, preferably at least90% or 95% sequence identity, and more preferably at least 97%, 98% or99% sequence identity. In certain embodiments, residue positions thatare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chainR group with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31(1994). Examples of groups of amino acids that have side chains withsimilar chemical properties include 1) aliphatic side chains: glycine,alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl sidechains: serine and threonine; 3) amide-containing side chains:asparagine and glutamine; 4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, andhistidine; 6) acidic side chains: aspartic acid and glutamic acid; and7) sulfur-containing side chains: cysteine and methionine. Conservativeamino acids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992), incorporated herein by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence identity for polypeptides is typically measured using sequenceanalysis software. Protein analysis software matches sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters as specified by the programs todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1 (University of Wisconsin, Wis.). Polypeptidesequences also can be compared using FASTA using default or recommendedparameters, see GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3)provides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences (Pearson, MethodsEnzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219(2000)). Another preferred algorithm when comparing a sequence of theinvention to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST, especially blastp ortblastn, using default parameters as supplied with the programs. See,e.g., Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul etal., Nucleic Acids Res. 25:3389-402 (1997).

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99% sequence identity to theantibodies or immunoglobulin molecules described herein. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat have related side chains. Genetically encoded amino acids aregenerally divided into families: (1) acidic=aspartate, glutamate; (2)basic=lysine, arginine, histidine; (3) non-polar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. More preferred families are: serine and threonineare an aliphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding function or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal., Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the antibodies described herein.

An antigen binding site is generally formed by the variable heavy (VH)and variable light (VL) immunoglobulin domains, with the antigen-bindinginterface formed by six surface polypeptide loops, termedcomplimentarity determining regions (CDRs). There are three CDRs in eachVH (HCDR1, HCDR2, HCDR3) and in each VL LCDR1, LCDR2, LCDR3), togetherwith framework regions (FRs).

Typically, a VH domain is paired with a V L domain to provide anantibody antigen-binding site, although a VH or VL domain alone may beused to bind antigen. The VH domain (see Table 4) may be paired with theVL domain (see Table 5), so that an antibody antigen-binding site isformed comprising both the VH and VL domains. Analogous embodiments areprovided for the other VH and VL domains disclosed herein. In otherembodiments, VH chains in Table 4 are paired with a heterologous VLdomain in Table 5. Light-chain promiscuity is well established in theart. Again, analogous embodiments are provided by the invention for theother VH and VL domains disclosed herein. Thus, the VH of the parent orof any of antibodies chain on Table 4 may be paired with the VL of theparent or of any of antibodies on Table 5 or other antibody.

An antigen binding site may comprise a set of H and/or L CDRs of theparent antibody or any of antibodies in Table 1 with as many as twenty,sixteen, ten, nine or fewer, e.g. one, two, three, four or five, aminoacid additions, substitutions, deletions, and/or insertions within thedisclosed set of H and/or L CDRs. Alternatively, an antigen binding sitemay comprise a set of H and/or L CDRs of the parent antibody or any ofantibodies Table 1 with as many as twenty, sixteen, ten, nine or fewer,e.g. one, two, three, four or five, amino acid substitutions within thedisclosed set of H and/or L CDRs. Such modifications may potentially bemade at any residue within the set of CDRs.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W.H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandonand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al., Nature 354:105 (1991), which are each incorporatedherein by reference.

A further aspect of the invention is an antibody molecule comprising aVH domain that has at least about 60, 70, 80, 85, 90, 95, 98 or about99% amino acid sequence identity with a VH domain of any of antibodylisted in Table 1, the appended sequence listing, an antibody describedherein or with an HCDR (e.g., HCDR1, HCDR2, or HCDR3) shown in Table 4or Table 4(a). The antibody molecule may optionally also comprise a VLdomain that has at least about 60, 70, 80, 85, 90, 95, 98 or about 99%amino acid sequence identity with a VL domain of any of antibody listedin Table 1, the appended sequence listing, an antibody described hereinor with an LCDR (e.g., LCDR1, LCDR2, or LCDR3) shown in Table 5.Algorithms that can be used to calculate % identity of two amino acidsequences comprise e.g. BLAST (Altschul et al. (1990) J. Mol. Biol. 215:405-410), FASTA (Pearson and Lipman (1988) PNAS USA 85: 2444-2448), orthe Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol.147: 195-197), e.g. employing default parameters.

Furthermore, variants of the VH and VL domains and CDRs of the presentinvention, including those for which amino acid sequences are set outherein, and which can be employed in targeted binding agents andantibodies for ERBB2 can be obtained by means of methods of sequencealteration or mutation and screening for antigen targeting with desiredcharacteristics. Examples of desired characteristics include but are notlimited to: increased binding affinity for antigen relative to knownantibodies which are specific for the antigen; increased neutralizationof an antigen activity relative to known antibodies which are specificfor the antigen if the activity is known; specified competitive abilitywith a known antibody or ligand to the antigen at a specific molarratio; ability to immunoprecipitate complex; ability to bind to aspecified epitope; linear epitope, e.g. peptide sequence identifiedusing peptide-binding scan as described herein, e.g. using peptidesscreened in linear and/or constrained conformation; conformationalepitope, formed by non-continuous residues; ability to modulate a newbiological activity of ERBb2, or downstream molecule. Such methods arealso provided herein.

Variants of antibody molecules disclosed herein may be produced and usedin the present invention. Following the lead of computational chemistryin applying multivariate data analysis techniques to thestructure/property-activity relationships (Wold, et al. Multivariatedata analysis in chemistry. Chemometries—Mathematics and Statistics inChemistry (Ed.: B. Kowalski), D. Reidel Publishing Company, Dordrecht,Holland, 1984) quantitative activity-property relationships ofantibodies can be derived using well-known mathematical techniques, suchas statistical regression, pattern recognition and classification(Norman et al. Applied Regression Analysis. Wiley-Interscience; 3rdedition (April 1998); Kandel, Abraham & Backer, Eric. Computer-AssistedReasoning in Cluster Analysis. Prentice Hall PTR, (May 11, 1995);Krzanowski, Wojtek. Principles of Multivariate Analysis: A User'sPerspective (Oxford Statistical Science Series, No 22 (Paper)). OxfordUniversity Press; (December 2000); Witten, Ian H. & Frank, Eibe. DataMining: Practical Machine Learning Tools and Techniques with JavaImplementations. Morgan Kaufmann; (Oct. 11, 1999); Denison David G. T.(Editor), Christopher C. Holmes, Bani K. Mallick, Adrian F. M. Smith.Bayesian Methods for Nonlinear Classification and Regression (WileySeries in Probability and Statistics). John Wiley & Sons; (July 2002);Chose, Arup K. & Viswanadhan, Vellarkad N. Combinatorial Library Designand Evaluation Principles, Software, Tools, and Applications in DrugDiscovery). The properties of antibodies can be derived from empiricaland theoretical models (for example, analysis of likely contact residuesor calculated physicochemical property) of antibody sequence, functionaland three-dimensional structures and these properties can be consideredsingly and in combination.

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain bindingspecificity. These predictions can be backed up by comparison of thepredictions to the output from lead optimization experiments. In astructural approach, a model can be created of the antibody moleculeusing any freely available or commercial package, such as WAM. A proteinvisualisation and analysis software package, such as Insight II(Accelrys, Inc.) or Deep View may then be used to evaluate possiblesubstitutions at each position in the CDR. This information may then beused to make substitutions likely to have a minimal or beneficial effecton activity.

The techniques required to make substitutions within amino acidsequences of CDRs, antibody VH or VL domains and/or targeted bindingagents generally are available in the art. Variant sequences may bemade, with substitutions that may or may not be predicted to have aminimal or beneficial effect on activity, and tested for ability to bindand/or neutralize a target and/or for any other desired property.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.

As used herein, the terms “label” or “labeled” refers to incorporationof another molecule on the antibody or targeted binding agent. In oneembodiment, the label is a detectable marker, e.g., incorporation of aradiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (e.g., streptavidincontaining a fluorescent marker or enzymatic activity that can bedetected by optical or colorimetric methods). In another embodiment, thelabel or marker can be therapeutic, e.g., a drug conjugate or toxin.Various methods of labeling polypeptides and glycoproteins are known inthe art and may be used. Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides(e.g., 3H, 14C, 15N, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I),fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic labels (e.g., horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase), chemiluminescent markers, biotinylgroups, predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags), magneticagents, such as gadolinium chelates, toxins such as pertussis toxin,taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

As used herein, a “targeted binding agent” is an agent, e.g. antibody,or binding fragment thereof, that preferentially binds to a target site.In one embodiment, the targeted binding agent is specific for only onetarget site. In other embodiments, the targeted binding agent isspecific for more than one target site. In one embodiment, the targetedbinding agent may be a monoclonal antibody and the target site may be anepitope. As described below, a targeted binding agent may comprise atleast one antigen binding domain of an antibody, wherein said domain isfused or contained within a heterologous protein scaffold, e.g. anon-antibody protein scaffold.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Human Anti-ErbB2 Antibodies and Characterization Thereof

In one embodiment, the invention provides anti-ErbB2 targeted bindingagent. In another embodiment, the invention provides anti-ErbB2antibodies. In some embodiments, the anti-ErbB2 antibodies are humanantibodies. In some embodiments, the invention provides anti-ErbB2antibodies that bind to human ErbB2 without the signal sequence, aminoacids 1-22 (Genbank ID: P04626) (SEQ ID NO:45). In some embodiments,human anti-ErbB2 antibodies are produced by immunizing a non-humantransgenic animal, e.g., a rodent, whose genome comprises humanimmunoglobulin genes so that the transgenic animal produces humanantibodies.

An anti-ErbB2 antibody of the invention can comprise a human kappa or ahuman lambda light chain or an amino acid sequence derived therefrom. Insome embodiments comprising a kappa light chain, the light chainvariable domain (VL) is encoded in part by a human VK1, VK2, or VK4family gene. In certain embodiments, the light chain utilizes a human VKA1, VK A2, VK B3, or VK L1 gene.

In various embodiments, the light chain variable domain utilizes a humanA2 gene and a human JK1 gene; a human L1 gene and a human JK5 gene; ahuman B3 gene and a human JK3 gene; or a human A1 gene and a human JK4gene.

In some embodiments, the VL of the ErbB2 antibody comprises one or moreamino acid substitutions relative to the germline amino acid sequence ofthe human gene. In some embodiments, the VL of the anti-ErbB2 antibodycomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acidsubstitutions relative to the germline amino acid sequence. In someembodiments, the VL of the anti-ErbB2 antibody comprises 0, 1, or 2amino acid insertions relative to the germline amino acid sequence. Insome embodiments, one or more of those substitutions from germline is inthe CDR regions of the light chain. In some embodiments, the amino acidsubstitutions relative to germline are at one or more of the samepositions as the substitutions relative to germline in any one or moreof the VL of antibodies 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3. For example, the VL of ananti-ErbB2 antibody of the invention may contain one or more amino acidsubstitutions compared to germline found in the VL of antibody 1.14, orthere may be one or more of the amino acid substitutions compared togermline found in the VL of antibody 1.18. In some embodiments, theamino acid changes are at one or more of the same positions, but involvea different substitution than in the reference antibody.

In some embodiments, amino acid changes relative to germline occur atone or more of the same positions as in any of the VL of antibodies1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39,1.24 and 1.71.3, but the changes may represent conservative amino acidsubstitutions at such position(s) relative to the amino acid in thereference antibody. For example, if a particular position in one ofthese antibodies is changed relative to germline and is glutamate, onemay substitute aspartate at that position. Similarly, if an amino acidsubstitution compared to germline is serine, one may conservativelysubstitute threonine for serine at that position. Conservative aminoacid substitutions are discussed supra.

In some embodiments, the light chain of the human anti-ErbB2 antibodycomprises the VL amino acid sequence of antibody 1.44.1 (SEQ ID NO:4),1.140 (SEQ ID NO:8), 1.4-3.1 (SEQ ID NO:12), 1.1-4.1 (SEQ ID NO:16),1.100.1 (SEQ ID NO:20), 1.9-6.2 (SEQ ID NO:24), 1.1-8.1 (SEQ ID NO:28),1.2-0.1 (SEQ ID NO:32), 1.3-9.1 (SEQ ID NO:36), 1.2-4.3 (SEQ ID NO:40),1.7-1.3 (SEQ ID NO:44) or said amino acid sequence having up to 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 conservative amino acidsubstitutions and/or a total of up to 3 non-conservative amino acidsubstitutions.

In certain embodiments, the light chain of the anti-ErbB2 antibodycomprises the light chain CDR1, CDR2 and CDR3 regions of an antibodycomprising the amino acid sequence of the VL region of an antibodyselected from antibody 1.44.1 (SEQ ID NO:4), 1.140 (SEQ ID NO:8),1.4-3.1 (SEQ ID NO:12), 1.1-4.1 (SEQ ID NO:16), 1.100.1 (SEQ ID NO:20),1.9-6.2 (SEQ ID NO:24), 1.1-8.1 (SEQ ID NO:28), 1.2-0.1 (SEQ ID NO:32),1.3-9.1 (SEQ ID NO:36), 1.2-4.3 (SEQ ID NO:40), 1.7-1.3 (SEQ ID NO:44)or said CDR regions each having less than 4 or less than 3 conservativeamino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions.

With regard to the heavy chain, in some embodiments, the variable domain(VH) is encoded in part by a human VH3 or VH4 family gene. In certainembodiments, the heavy chain VH utilizes a human VH3-21, VH3-13, VH4-31,or VH3-7 gene. In various embodiments, the heavy chain VH utilizes ahuman VH3-21 gene, a human D5-24 gene and a human JH4B gene. In otherembodiments, the heavy chain VH utilizes a human VH3-7 gene, and a humanJH6; a human VH4-31 gene, a human D3-10 gene and a human JH6B gene; or ahuman VH3-13 gene, a human D6-19 gene and a human JH6B gene. In someembodiments, the VH sequence of the anti-ErbB2 antibody contains one ormore amino acid substitutions, deletions or insertions (additions)relative to the germline amino acid sequence.

In some embodiments, the variable domain of the heavy chain comprises 1,2, 3, 4, 5, 6, or 7 mutations from the germline amino acid sequence; 0,1, 2, or 3 of which maybe substitutions. In some embodiments, thevariable domain of the heavy chain comprises 0, 1, 2, or 3 additionscompared to the germline amino acid sequence. In some embodiments, themutation(s) are non-conservative substitutions compared to the germlineamino acid sequence. In some embodiments, the mutations are in the CDRregions of the heavy chain. In some embodiments, the amino acid changesare made at one or more of the same positions as the mutations fromgermline in any one or more of the VH of antibodies 1.14.1, 1.18.1,1.19, 1.20.1, 1.22.1, 1.22.2, 1.24.3, 1.41, 1.43.1, 143.2, 1.44.1,1.39.1, 1.71.1, 1.71.3, 1.96.2, 1.99, 1.100.1, 1.104, 1.107, 1.124,1.128, 1.140.1, or 1.148. In other embodiments, the amino acid changesare at one or more of the same positions but involve a differentmutation than in the reference antibody.

In some embodiments, the heavy chain comprises the VH amino acidsequence of antibody 1.44.1 (SEQ ID NO:2), 1.140.1 (SEQ ID NO:6),1.4-3.1 (SEQ ID NO:10), 1.14.1 (SEQ ID NO:14), 1.100.1 (SEQ ID NO:18),1.9-6.2 (SEQ ID NO:22), 1.1-8.1 (SEQ ID NO:26), 1.2-0.1 (SEQ ID NO:30),1.3-9.1 (SEQ ID NO:34), 1.2-4.3 (SEQ ID NO:38), 1.7-1.3 (SEQ ID NO:42);or said VH amino acid sequence having up to 1, 2, 3, 4, 6, 8, orconservative amino acid substitutions and/or a total of up to 3non-conservative amino acid substitutions.

In some embodiments, the heavy chain comprises the heavy chain CDR1,CDR2 and CDR3 regions of antibody 1.14.1, 1.18.1, 1.19, 1.20.1, 1.22.1,1.22.2, 1.24.3, 1.41, 1.43.1, 143.2, 1.44.1, 1.39.1, 1.71.1, 1.71.3,1.96.2, 1.99, 1.100.1, 1.104, 1.107, 1.124, 1.128, 1.140.1, or 1.148 orsaid CDR regions each having less than 5, less than 4, less than 3, orless than 2 conservative amino acid substitutions and/or a total ofthree or fewer non-conservative amino acid substitutions.

In another embodiment, the antibody comprises the light chain asdisclosed above and a heavy chain as disclosed above. In a furtherembodiment, the light chain CDRs and the heavy chain CDRs are from thesame antibody.

One type of amino acid substitution that may be made is to change one ormore cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. In oneembodiment, there is a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant domain of an antibody. In some embodiments,the cysteine is canonical.

Another type of amino acid substitution that may be made is to changeany potential proteolytic sites in the antibody. Such sites may occur ina CDR or framework region of a variable domain or in the constant domainof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of any heterogeneity in theantibody product and thus increase its homogeneity. Another type ofamino acid substitution is to eliminate asparagine-glycine pairs, whichform potential deamidation sites, by altering one or both of theresidues. Still another type of amino acid substitution is at methionineresidues to eliminate oxidation sites.

In some embodiments, the C-terminal lysine of the heavy chain of theanti ErbB2 antibody of the invention is cleaved. In various embodimentsof the invention, the heavy and light chains of the anti-ErbB2antibodies may optionally include a signal sequence.

In one aspect, the invention relates to inhibitory human anti-ErbB2monoclonal antibodies and the hybridoma cell lines that produce them.Table 1 lists the sequence identifiers (SEQ ID NO:) of the nucleic acidsencoding the variable domain of the heavy and light chains, and thecorresponding deduced amino acid sequences.

TABLE 1 HUMAN ANTI-ErbB2 ANTIBODIES SEQUENCE IDENTIFIER (SEQ ID NO:)Variable Domain-Comprising Portion Monoclonal Heavy Light Antibody DNAProtein DNA Protein 1.44.1 1 2 3 4 1.140 5 6 7 8 1.43 9 10 11 12 1.14.113 14 15 16 1.100.1 17 18 19 20 1.96 21 22 23 24 1.18.1 25 26 27 28 1.2029 30 31 32 1.39 33 34 35 36 1.24 37 38 39 40 1.71.3 41 42 43 44Class and Subclass of Anti-ErbB2 Antibodies

The class and subclass of anti-ErbB2 antibodies may be determined by anymethod known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies arecommercially available. The class and subclass can be determined byELISA, or Western Blot as well as other techniques. Alternatively, theclass and subclass may be determined by sequencing all or a portion ofthe constant domains of the heavy and/or light chains of the antibodies,comparing their amino acid sequences to the known amino acid sequencesof various class and subclasses of immunoglobulins, and determining theclass and subclass of the antibodies.

In some embodiments, the anti-ErbB2 antibody is a monoclonal antibody.The anti-ErbB2 antibody can be an IgG, an IgM, an IgE, an IgA, or an IgDmolecule. In another embodiment, the anti-ErbB2 antibody is an IgG andis an IgG1, IgG2, IgG3, IgG4 subclass. In another preferred embodiment,the antibody is subclass IgG2 (see Kabat et al., (1991) Sequences ofProteins of Immunological Interest, 5th edn. US Department of Health andHuman Services, Washington, D.C.).

Binding Affinity of Anti-ErbB2 Targeted Binding Agents and Antibodies toErbB2

In some embodiments of the invention, targeted binding agents and/oranti-ErbB2 antibodies bind to ErbB2 with high affinity. In someembodiments, the targeted binding agent and/or anti-ErbB2 antibody bindsto ErbB2 with a K_(D) of 13.5 10⁻⁹M or less using high resolutionbiocore analysis. In still other embodiments, the targeted binding agentand/or antibody binds to ErbB2 with a K_(D) of 13×10⁻⁹M, 13×10⁻⁹M,11×10⁻⁹M, 12×10⁻⁹M, 10×10⁻⁹M, 5×10⁻⁹M, 3×10⁻⁹, 2×10⁻⁹, 1×10⁻⁹M or5×10⁻¹° M or less using high resolution biocore analysis. In certainembodiments, the targeted binding agent and/or antibody binds to ErbB2with substantially the same K_(D) as an antibody selected from 1.44.1,1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39,1.24 and 1.71.3. In still another preferred embodiment, the targetedbinding agent and/or antibody binds to ErbB2 with substantially the sameK_(D) as an antibody that comprises a heavy chain variable domain havingthe amino acid sequence of the V_(H) region found in SEQ ID NO: 2, 6,10, 14, 18, 22, 26, 30, 34, 38, or 42, a light chain variable domainhaving the amino acid sequence of the V_(L) region found in SEQ ID NO:4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, or both. In another preferredembodiment, the antibody binds to ErbB2 with substantially the sameK_(D) as a targeted binding agent and/or an antibody that comprises theCDR regions of a light chain variable domain having the amino acidsequence of the V_(L) region found in SEQ ID NO: 4, 8, 12, 16, 20, 24,28, 32, 36, 40, 44 or that comprises the CDR regions of a heavy chainvariable domain having the amino acid sequence the V_(H) region found inSEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, or 42, or both.

In some embodiments, the targeted binding agent and/or anti-ErbB2antibody has a low dissociation rate constant (k_(off)) In someembodiments, the targeted binding agents and/or anti-ErbB2 antibody hasa k_(off) of 1.0×10⁻³ s-1 or lower or a k_(off) of 5.0×10⁻⁴s-1 or lower.In other preferred embodiments, the antibody binds to ErbB2 with ak_(off) of 2×10⁻⁴ s-1 or lower. In some embodiments, the k_(off) issubstantially the same as an antibody described herein, including anantibody selected from 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3. In some embodiments, thetargeted binding agent and/or antibody binds to ErbB2 with substantiallythe same k_(off) as an antibody that comprises the CDR regions of aheavy chain, or the CDR regions of a light chain from an antibodyselected from 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20,1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,1.20, 1.39, 1.24 and 1.71.3, or both. In some embodiments, the targetedbinding agents and/or antibody binds to ErbB2 with substantially thesame k_(off) as an antibody that comprises a heavy chain variable domainhaving the amino acid sequence of the V_(H) region found in SEQ ID NO:2, 6, 10, 14, 18, 22, 26, 30, 34, 38, or 42, a light chain variabledomain having the amino acid sequence of the V_(L) region found in SEQID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 or both.

The binding affinity and dissociation rate of a targeted binding agentand an anti-ErbB2 antibody to ErbB2 can be determined by methods knownin the art. The binding affinity can be measured by ELISAs, RIAs, flowcytometry, surface plasmon resonance, such as BIACORE™. The dissociaterate can be measured by surface plasmon resonance. Preferably, thebinding affinity and dissociation rate is measured by surface plasmonresonance. More preferably, the binding affinity and dissociation rateare measured using BIACORE™. One can determine whether an antibody hassubstantially the same K_(D) as an anti-ErbB2 antibody by using methodsknown in the art. Example 12 exemplifies a method for determiningaffinity constants of anti-ErbB2 monoclonal antibodies by flowcytometry.

Identification of ErbB2 Epitopes Recognized by Anti-ErbB2 Antibodies

The invention provides targeted binding agents and/or human anti-ErbB2monoclonal antibodies that binds to ErbB2 and competes or competes withand/or binds the same epitope as: (a) an antibody selected from 1.44.1,1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39,1.24 and 1.71.3; (b) an antibody that comprises a heavy chain variabledomain having an amino acid sequence of the variable domain found in SEQID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, or 42, (c) an antibody thatcomprises a light chain variable domain having an amino acid sequence ofthe variable domain of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40,44, or (d) an antibody that comprises both a heavy chain variable domainas defined in (b) and a light chain variable domain as defined in (c).If two antibodies reciprocally compete with each other for binding toErbB2, they are said to compete.

One can determine whether a targeted binding agent and/or an antibodybinds to the same epitope or competes for binding with an anti-ErbB2antibody by using methods known in the art. In one embodiment, oneallows the anti-ErbB2 antibody of the invention to bind to ErbB2 undersaturating conditions and then measures the ability of the test antibodyto bind to ErbB2. If the test antibody is able to bind to ErbB2 at thesame time as the anti-ErbB2 antibody, then the test antibody binds to adifferent epitope as the anti-ErbB2 antibody. However, if the testantibody is not able to bind to ErbB2 at the same time, then the testantibody binds to the same epitope, an overlapping epitope, or anepitope that is in close proximity to the epitope bound by the humananti-ErbB2 antibody. This experiment can be performed using ELISA, RIA,BIACORE™, or flow cytometry.

To test whether a targeted binding agent and/or an anti-ErbB2 antibodycompetes with another anti-ErbB2 antibody, one may use the competitionmethod described above in two directions i.e. determining if thereference antibody blocks the test antibody and vice versa. In anotherembodiment, the experiment is performed using ELISA. Methods ofdetermining K_(D) are discussed further below.

Inhibition of ErbB2 Activity by Anti-ErbB2 Antibody

In various embodiments, the invention provides targeted binding agentsand/or anti-ErbB2 antibodies that inhibits signaling via ErbB2. In oneembodiment, the targeted binding agent and/or an anti-ErbB2 antibodyinhibits ligand-induced signaling of ErbB2. In one embodiment, thetargeted binding agent and/or an anti-ErbB2 antibody inhibitsligand-induced signaling of ErbB2 without blocking the binding of theligand to ErbB2. In another embodiment, the ErbB2 is human. In anotherembodiment, the anti-ErbB2 antibody is a human antibody. In someembodiments the ligand is Heregulin-β. The EC₅₀ of the targeted bindingagent and/or anti-ErbB2 antibody can be measured by detecting thebinding of the antibody to the antigen in a direct binding assaymonitored by ELISA or RIA, or via cell-based assays such as thosedescribed below. In one embodiment, the targeted binding agent and/orantibody or portion thereof inhibits the ligand-induced signaling viathe ErbB2 receptor with an EC₅₀ of no more than 50 ng/ml, preferably nomore than 25 ng/ml, more preferably no more than 10 ng/ml, even morepreferably no more than 5 ng/ml. In some embodiments, the targetedbinding agent and/or anti-ErbB2 antibody inhibits ligand-inducedsignaling of ErbB2 by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. Measuring inhibition canbe accomplished by any means known in the art.

In another embodiment, the invention provides a targeted binding agentand/or an anti-ErbB2 antibody that inhibits phosphorylation of ErbB2. Invarious embodiments, the EC₅₀ of the targeted binding agent and/orantibody is no more than 50 ng/ml, preferably no more than 25 ng/ml,more preferably no more than 10 ng/ml, even more preferably no more than5 ng/ml. In some embodiments, the targeted binding agents and/oranti-ErbB2 antibody inhibits phosphorylation of ErbB2 by at least 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or 100%. Examples 4, 9, and 11 exemplify an ErbB2 phosphorylationassay.

In another embodiment, the invention provides a targeted binding agentand/or an anti-ErbB2 antibody that inhibits activation of the MAPKpathway. In various embodiments, the EC₅₀ of the targeted binding agentand/or antibody is no more than 50 ng/ml, preferably no more than 25ng/ml, more preferably no more than 10 ng/ml, even more preferably nomore than 5 ng/ml. In some embodiments, the targeted binding agentand/or anti-ErbB2 antibody inhibits phosphorylation of ErbB2 by at least20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 100%. Assays for monitoring MAPK pathway activation in acell are known in the art (see U.S. Patent Application Nos. 20030186382,and 20030096333, herein incorporated by reference in their entiretiesfor all purposes).

In another embodiment, the invention provides a targeted binding agentand/or an anti-ErbB2 antibody that modulates the activity of thep38-TSP-1 pathway. In some embodiments, the targeted binding agentand/or antibody activates the p38-TSP-1 pathway. In other embodiments,the targeted binding agent and/or antibody inhibits the p38-TSP-1pathway. In various embodiments, the EC₅₀ of the targeted binding agentand/or antibody is no more than 50 ng/ml, preferably no more than 25ng/ml, more preferably no more than 10 ng/ml, even more preferably nomore than 5 ng/ml. In some embodiments, the targeted binding agentand/or anti-ErbB2 antibody inhibits phosphorylation of ErbB2 by at least20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 100%. Assays for monitoring p38-TSP-1 pathway activation ina cell are known in the art (see U.S. Patent Application Nos.20060089393 and 20020103253, herein incorporated by reference in theirentireties for all purposes).

In another embodiment, the invention provides a targeted binding agentand/or an anti-ErbB2 antibody that inhibits activation of the PI3Kpathway. In various embodiments, the EC₅₀ of the targeted binding agentand/or antibody is no more than 50 ng/ml, preferably no more than 25ng/ml, more preferably no more than 10 ng/ml, even more preferably nomore than 5 ng/ml. In some embodiments, the targeted binding agentand/or anti-ErbB2 antibody inhibits phosphorylation of ErbB2 by at least20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 100%. Assays for monitoring PI3K pathway activation in acell are known in the art (see U.S. Patent Application Nos. 20020037276and 20040176385, herein incorporated by reference in their entiretiesfor all purposes).

In another embodiment, the invention provides a targeted binding agentand/or an anti-ErbB2 antibody that inhibits inhibition of CDC2. Invarious embodiments, the EC₅₀ of the targeted binding agent and/or theantibody is no more than 50 ng/ml, preferably no more than 25 ng/ml,more preferably no more than 10 ng/ml, even more preferably no more than5 ng/ml. In some embodiments, the targeted binding agent and/oranti-ErbB2 antibody inhibits phosphorylation of ErbB2 by at least 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or 100%. Assays for monitoring inhibition of CDC2 in a cell areknown in the art (see U.S. Patent Application Nos. 20030225098 and20040110775, herein incorporated by reference in their entireties forall purposes).

Inhibition of Cell Proliferation with Anti-ErbB2 Antibodies

According to some embodiments, the invention provides a targed bindingagent and/or an anti-ErbB2 antibody that inhibits the proliferation ofcancer or transformed cells in vivo or in vitro or both. In anotherembodiment, the targed binding agent and/or anti-ErbB2 antibody inhibitsproliferation by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 100%. In one embodiment, the phosphorylationis measured at least 1 day after the animals have started treatment withthe antibody and proliferation is measured 3 days after the animals havestarted treatment with the antibody. In another embodiment, theinhibition is measured at least one hour after the animals have startedtreatment with the antibody. In various embodiments, the EC₅₀ of thetarged binding agent and/or antibody, as measured by cell titer or aproliferation marker, is no more than 3.5 μg/ml, preferably no more than300 ng/ml, more preferably no more than 100 ng/ml, even more preferablyno more than 50 ng/ml. Examples 9 and 10 exemplify proliferation assays.

Species and Molecular Selectivity

In another aspect of the invention, the targeted binding agents and/oranti-ErbB2 antibodies demonstrate both species and molecularselectivity. In some embodiments, the targed binding agent and/oranti-ErbB2 antibody binds to human (SEQ ID NO:45) and cynomologus ErbB2.Following the teachings of the specification, one may determine thespecies selectivity for the targed binding agent and/or anti-ErbB2antibody using methods well known in the art. For instance, one maydetermine the species selectivity using Western blot, flow cytometry,ELISA, immunoprecipitation or RIA. In another embodiment, one maydetermine the species selectivity using flow cytometry.

In some embodiments, the targed binding agent and/or anti-ErbB2 antibodydoes not exhibit any appreciable specific binding to any other proteinother than ErbB2. One can determine the selectivity of the targedbinding agent and/or anti-ErbB2 antibody for ErbB2 using methods wellknown in the art following the teachings of the specification. Forinstance one can determine the selectivity using Western blot, flowcytometry, ELISA, immunoprecipitation or RIA.

Methods of Producing Antibodies and Antibody Producing Cell Lines

In some embodiments, human antibodies are produced by immunizing anon-human, transgenic animal comprising within its genome some or all ofhuman immunoglobulin heavy chain and light chain loci with an ErbB2antigen. In another embodiment, the non-human animal is a XENOMOUSE™animal. (Amgen Fremont, Inc., Fremont, Calif.).

XENOMOUSE™ mice are engineered mouse strains that comprise largefragments of human immunoglobulin heavy chain and light chain loci andare deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364,6,162,963 and 6,150,584. See also WO 91/10741, WO 94/02602, WO 96/34096,WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO99/53049, WO 00/09560, and WO 00/037504.

In another aspect, the invention provides a method for making anti-ErbB2antibodies from non-human, non-mouse animals by immunizing non-humantransgenic animals that comprise human immunoglobulin loci with an ErbB2antigen. One can produce such animals using the methods described in theabove-cited documents. The methods disclosed in these documents can bemodified as described in U.S. Pat. No. 5,994,619, which is herebyincorporated by reference. U.S. Pat. No. 5,994,619 describes methods forproducing novel cultured inner cell mass (CICM) cells and cell lines,derived from pigs and cows, and transgenic CICM cells into whichheterologous DNA has been inserted. CICM transgenic cells can be used toproduce cloned transgenic embryos, fetuses, and offspring. The '619patent also describes methods of producing transgenic animals that arecapable of transmitting the heterologous DNA to their progeny. Inpreferred embodiments of the current invention, the non-human animalsare mammals, particularly rats, sheep, pigs, goats, cattle or horses.

XENOMOUSE™ mice produce an adult-like human repertoire of fully humanantibodies and generate antigen-specific human antibodies. In someembodiments, the XENOMOUSE™ mice contain approximately 80% of the humanantibody V gene repertoire through introduction of germlineconfiguration fragments of the human heavy chain loci and kappa lightchain loci in yeast artificial chromosome (YAC). In other embodiments,XENOMOUSE™ mice further contain approximately all of the human lambdalight chain locus. See Mendez et al., Nature Genetics 15:146-156 (1997),Green and Jakobovits, J. Exp. Med. 188:483-495 (1998), and WO 98/24893,the disclosures of which are hereby incorporated by reference. In otherembodiments, the antibodies are produced in human trans-chromosomic mice(see WO 02/43478 and WO 02/092812, hereby incorporated by reference).

In some embodiments, the non-human animal comprising humanimmunoglobulin genes are animals that have a human immunoglobulin“minilocus”. In the minilocus approach, an exogenous Ig locus ismimicked through the inclusion of individual genes from the Ig locus.Thus, one or more V_(H) genes, one or more D_(H) genes, one or moreJ_(H) genes, a mu constant domain, and a second constant domain(preferably a gamma constant domain) are formed into a construct forinsertion into an animal. This approach is described, inter cilia, inU.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205,5,721,367, 5,789,215, and 5,643,763, hereby incorporated by reference.

In another aspect, the invention provides a method for making humanizedanti-ErbB2 antibodies. In some embodiments, non-human animals areimmunized with an ErbB2 antigen as described below under conditions thatpermit antibody production. Antibody-producing cells are isolated fromthe animals, fused with myelomas to produce hybridomas, and nucleicacids encoding the heavy and light chains of an anti-ErbB2 antibody ofinterest are isolated. These nucleic acids are subsequently engineeredusing techniques known to those of skill in the art and as describedfurther below to reduce the amount of non-human sequence, i.e., tohumanize the antibody to reduce the immune response in humans

In some embodiments, the ErbB2 antigen is isolated and/or purifiedErbB2. In another embodiment, the ErbB2 antigen is human ErbB2. In someembodiments, the ErbB2 antigen is a fragment of ErbB2. In someembodiments, the ErbB2 fragment is an extracellular domain of ErbB2. Insome embodiments, the ErbB2 fragment is an extracellular loop of ErbB2(see Cho et al., Nature. 2003 Feb. 13; 421(6924):756-60, herebyincorporated by reference). In some embodiments, the ErbB2 fragmentcomprises at least one epitope of ErbB2. In other embodiments, the ErbB2antigen is a cell that expresses or overexpresses ErbB2, or animmunogenic fragment thereof, on its surface. In some embodiments, theErbB2 antigen is an ErbB2 fusion protein. In some embodiments, the ErbB2is a synthetic peptide immunogen.

Immunization of animals can be by any method known in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619.In another embodiment, the ErbB2 antigen is administered with anadjuvant to stimulate the immune response. Exemplary adjuvants includecomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. Preferably, if a polypeptide is being administered, theimmunization schedule will involve two or more administrations of thepolypeptide, spread out over several weeks. Example 1 exemplifies amethod for producing anti-ErbB2 monoclonal antibodies in XenoMouse™mice.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with an ErbB2 antigen, antibodies and/orantibody-producing cells can be obtained from the animal. In someembodiments, anti-ErbB2 antibody-containing serum is obtained from theanimal by bleeding or sacrificing the animal. The serum may be used asit is obtained from the animal, an immunoglobulin fraction may beobtained from the serum, or the anti-ErbB2 antibodies may be purifiedfrom the serum.

In some embodiments, antibody-producing immortalized cell lines areprepared from cells isolated from the immunized animal. Afterimmunization, the animal is sacrificed and lymph node and/or splenic Bcells are immortalized by any means known in the art. Methods ofimmortalizing cells include, but are not limited to, transfecting themwith oncogenes, infecting them with an oncogenic virus and cultivatingthem under conditions that select for immortalized cells, subjectingthem to carcinogenic or mutating compounds, fusing them with animmortalized cell, e.g., a myeloma cell, and inactivating a tumorsuppressor gene. See, e.g., Harlow and Lane, supra. If fusion withmyeloma cells is used, the myeloma cells preferably do not secreteimmunoglobulin polypeptides (a non-secretory cell line). Immortalizedcells are screened using ErbB2, a portion thereof, or a cell expressingErbB2. In another embodiment, the initial screening is performed usingan enzyme-linked immunoassay (ELISA) or a radioimmunoassay. An exampleof ELISA screening is provided in WO 00/37504, incorporated herein byreference.

Anti-ErbB2 antibody-producing cells, e.g., hybridomas, are selected,cloned and further screened for desirable characteristics, includingrobust growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas can be expandedin vivo in syngeneic animals, in animals that lack an immune system,e.g., nude mice, or in cell culture in vitro. Methods of selecting,cloning and expanding hybridomas are well known to those of ordinaryskill in the art.

In one embodiment, the immunized animal is a non-human animal thatexpresses human immunoglobulin genes and the splenic B cells are fusedto a myeloma cell line from the same species as the non-human animal. Ina more preferred embodiment, the immunized animal is a XENOMOUSE™ mouseand the myeloma cell line is a non-secretory mouse myeloma. In an evenmore preferred embodiment, the myeloma cell line is P3-X63-Ag8.653(American Type Culture Collection). See, e.g., Example 2.

Thus, in one embodiment, the invention provides methods for producing acell line that produces a human monoclonal antibody or a fragmentthereof directed to ErbB2 comprising (a) immunizing a non-humantransgenic animal described herein with ErbB2, a portion of ErbB2 or acell or tissue expressing ErbB2; (b) allowing the transgenic animal tomount an immune response to ErbB2; (c) isolating antibody-producingcells from transgenic animal; (d) immortalizing the antibody-producingcells; (e) creating individual monoclonal populations of theimmortalized antibody-producing cells; and (f) screening theimmortalized antibody-producing cells to identify an antibody directedto ErbB2.

In another aspect, the invention provides hybridomas that produce ahuman anti-ErbB2 antibody. In another embodiment, the hybridomas aremouse hybridomas, as described above. In other embodiments, thehybridomas are produced in a non-human, non-mouse species such as rats,sheep, pigs, goats, cattle or horses. In another embodiment, thehybridomas are human hybridomas.

In one embodiment of the invention, antibody-producing cells areisolated and expressed in a host cell, for example myeloma cells. Inanother preferred embodiment, a transgenic animal is immunized withErbB2, primary cells, e.g., spleen or peripheral blood cells, areisolated from an immunized transgenic animal and individual cellsproducing antibodies specific for the desired antigen are identified.Polyadenylated mRNA from each individual cell is isolated and reversetranscription polymerase chain reaction (RT-PCR) is performed usingsense primers that anneal to variable region sequences, e.g., degenerateprimers that recognize most or all of the FR1 regions of human heavy andlight chain variable region genes and anti-sense primers that anneal toconstant or joining region sequences. cDNAs of the heavy and light chainvariable domains are then cloned and expressed in any suitable host cellas chimeric antibodies with respective immunoglobulin constant regions,such as the heavy chain and K or), light chain constant domains. SeeBabcook, J. S. et al., Proc. Natl. Acad. Sci. USA 93:7843-48, 1996,incorporated herein by reference. Anti ErbB2 antibodies may then beidentified and isolated as described herein.

In another embodiment, phage display techniques can be used to providelibraries containing a repertoire of antibodies with varying affinitiesfor ErbB2. For production of such repertoires, it is unnecessary toimmortalize the B cells from the immunized animal. Rather, the primary Bcells can be used directly as a source of DNA. The mixture of cDNAsobtained from B cell, e.g., derived from blood or spleens, is used toprepare an expression library, for example, a human phage displaylibrary transfected into E. coli. The resulting cells are tested forimmunoreactivity to ErbB2. Techniques for the identification of highaffinity human antibodies from such libraries are described by Griffithset al., EMBO J., 13:3245-3260 (1994); Nissim et al., ibid, pp. 692-698and by Griffiths et al., ibid, 12:725-734, which are incorporated byreference. Ultimately, clones from the library are identified thatproduce binding affinities of a desired magnitude for the antigen andthe DNA encoding the product responsible for such binding is recoveredand manipulated for standard recombinant expression. Phage displaylibraries may also be constructed using previously manipulatednucleotide sequences and screened in a similar fashion. In general, thecDNAs encoding heavy and light chains are independently supplied orlinked to form Fv analogs for production in the phage library. Incertain embodiments, chain shuffling may be utilized (see Kang et al.,PNAS (1991) December 15; 88(24):11120-3, hereby incorporated byreference).

The phage library is then screened for the antibodies with the highestaffinities for ErbB2 and the genetic material recovered from theappropriate clone. Further rounds of screening can increase affinity ofthe original antibody isolated.

Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of MakingAntibodies

Nucleic Acids

The present invention also encompasses nucleic acid molecules encodinganti-ErbB2 antibodies or antigen-binding portions thereof. In someembodiments, different nucleic acid molecules encode a heavy chain and alight chain of an anti-ErbB2 immunoglobulin. In other embodiments, thesame nucleic acid molecule encodes a heavy chain and a light chain of ananti-ErbB2 immunoglobulin. In one embodiment, the nucleic acid encodesan ErbB2 antibody of the invention.

In some embodiments, the nucleic acid molecule encoding the variabledomain of the light chain (V_(L)) utilizes a human Vκ B3, Vκ L1, Vκ A2,or Vκ A1 gene, and a Jκ1, Jκ3, Jκ4 or J_(k)S gene with or withoutmutation from the germline.

In some embodiments, the nucleic acid molecule encoding the light chain,encodes an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13 substitutions and/or 0, 1, or 2 insertions relative to thegermline amino acid sequence(s). In some embodiments, the nucleic acidmolecule comprises a nucleotide sequence that encodes a V_(L) amino acidsequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13conservative amino acid substitutions and/or a total of up to 3non-conservative substitutions compared to germline V_(K) and J_(K)sequences. Substitutions may be in the CDR regions, the frameworkregions, or in the constant domain.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence comprising one or more variants compared to germlinesequence that are identical to the variations found in the V_(L) of anyone of the antibodies 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3.

In some embodiments, the nucleic acid molecule encodes at least threeamino acid substitutions compared to the germline sequence found in theV_(L) of one of the antibodies 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1,1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the V_(L) amino acid sequence of monoclonalantibody 1.44.1 (SEQ ID NO:4), 1.140 (SEQ ID NO:8), 1.4-3.1 (SEQ IDNO:12), 1.1-4.1 (SEQ ID NO:16), 1.100.1 (SEQ ID NO:20), 1.9-6.2 (SEQ IDNO:24), 1.1-8.1 (SEQ ID NO:28), 1.2-0.1 (SEQ ID NO:32), 1.3-9.1 (SEQ IDNO:36), 1.2-4.3 (SEQ ID NO:40), 1.7-1.3 (SEQ ID NO:44), or a variant orportion thereof. In some embodiments, the nucleic acid encodes an aminoacid sequence comprising the light chain CDRs of one of saidabove-listed antibodies.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one of SEQ ID NO: 4, 8,12, 16, 20, 24, 28, 32, 36, 40, or 44. In some preferred embodiments,the nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, or a portion thereof.

In some embodiments, the nucleic acid encodes the amino acid sequence ofthe light chain CDRs of said antibody.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to a V_(L) amino acid sequence of a V_(L) region of anyone of antibodies 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1,1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96,1.18.1, 1.20, 1.39, 1.24 and 1.71.3, or an amino acid sequence of anyone of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, or 44. Nucleicacid molecules of the invention include nucleic acids that hybridizeunder highly stringent conditions, such as those described above, to anucleotide sequence encoding the amino acid sequence of a V_(L) regionfound in SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, or thathas the nucleotide sequence of a nucleic acid molecule encoding theV_(L) region found in SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39,or 43.

In another embodiment, the nucleic acid encodes a full-length lightchain of an antibody selected from 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.31.44.1, 1.140, 1.43, 1.14.1,1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3.

In another preferred embodiment, the nucleic acid molecule encodes thevariable domain of a heavy chain (V_(H)) that comprises a human V_(H)3-21, a human V_(H) 3-7, a human V_(H) 4-31, or a human V_(H) 3-13 genesequence or a sequence derived therefrom. In various embodiments, thenucleic acid molecule utilizes a human V_(H)3-7 gene, and a humanJ_(H)6; a human V_(H)4-31 gene, a human D3-10 gene and a human J_(H)6Bgene; or a human V_(H)3-13 gene, a human D6-19 gene and a human J_(H)6Bgene.

In some embodiments, the nucleic acid molecule encodes an amino acidsequence comprising 1, 2, 3, 4, 5, 6, or 7 mutations compared to thegermline amino acid sequence of the human V, D and J genes; 0, 1, 2, or3 of which maybe substitutions. In some embodiments, said mutations arein the V_(H) region. In some embodiments, said mutations are in the CDRregions.

In some embodiments, the nucleic acid molecule encodes one or more aminoacid mutations compared to the germline sequence that are identical toamino acid mutations found in the V_(H) of monoclonal antibody 1.14.1,1.18.1, 1.19, 1.20.1, 1.22.1, 1.22.2, 1.24.3, 1.41, 1.43.1, 143.2,1.44.1, 1.39.1, 1.71.1, 1.71.3, 1.96.2, 1.99, 1.100.1, 1.104, 1.107,1.124, 1.128, 1.140.1, or 1.148. In some embodiments, the nucleic acidencodes at least three amino acid mutations compared to the germlinesequences that are identical to at least three amino acid mutationsfound in one of the above-listed monoclonal antibodies.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes at least a portion of the V_(H) amino acidsequence of a antibody selected from 1.44.1 (SEQ ID NO:2), 1.140.1 (SEQID NO:6), 1.4-3.1 (SEQ ID NO:10), 1.1-4.1 (SEQ ID NO:14), 1.100.1 (SEQID NO:18), 1.9-6.2 (SEQ ID NO:22), 1.1-8.1 (SEQ ID NO:26), 1.2-0.1 (SEQID NO:30), 1.3-9.1 (SEQ ID NO:34), 1.2-4.3 (SEQ ID NO:38), 1.7-1.3 (SEQID NO:42), a variant thereof, or said sequence having conservative aminoacid mutations and/or a total of three or fewer non-conservative aminoacid substitutions. In various embodiments the sequence encodes one ormore CDR regions, preferably a CDR3 region, all three CDR regions, orthe entire V_(H) region.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one of SEQ ID NO: 2, 6,10, 14, 18, 22, 26, 30, 34, 38, and 42. In some preferred embodiments,the nucleic acid molecule comprises at least a portion of the nucleotidesequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, or 41. Insome embodiments, said portion encodes the V_(H) region, a CDR3 regionor all three CDR regions.

In some embodiments, the nucleic acid molecule encodes a V_(H) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the V_(H) amino acid sequence of any one of SEQ ID NO:2, 6, 10, 14, 18, 22, 26, 30, 34, 38, or 42. Nucleic acid molecules ofthe invention include nucleic acids that hybridize under highlystringent conditions, such as those described above, to a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 1, 5, 9, 13, 17,21, 25, 29, 33, 37, or 41, or to a V_(H) region thereof, or that has thenucleotide sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37,or 41 or that encodes a V_(H) region thereof.

In another embodiment, the nucleic acid encodes a full-length heavychain of an antibody selected from 1.44.1, 1.140, 1.43, 1.14.1, 1.100.1,1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3.

A nucleic acid molecule encoding the heavy or light chain of ananti-ErbB2 antibody or portions thereof can be isolated from any sourcethat produces such antibody. In various embodiments, the nucleic acidmolecules are isolated from a B cell isolated from an animal immunizedwith ErbB2, from an immortalized cell derived from such a B cell thatexpresses an anti-ErbB2 antibody, or from a bacteriophage. Methods ofisolating mRNA encoding an antibody are well-known in the art. See,e.g., Sambrook et al. The mRNA may be used to produce cDNA for use inthe polymerase chain reaction (PCR) or cDNA cloning of antibody genes.In one embodiment, the nucleic acid molecule is isolated from ahybridoma that has as one of its fusion partners a humanimmunoglobulin-producing cell from a non-human transgenic animal. Inanother embodiment, the human immunoglobulin producing cell is isolatedfrom a XENOMOUSE™ animal. In another embodiment, the humanimmunoglobulin-producing cell is from a non-human, non-mouse transgenicanimal, as described above. In another embodiment, the nucleic acid isisolated from a non-human, non-transgenic animal. The nucleic acidmolecules isolated from a non-human, non-transgenic animal may be used,e.g., for humanized antibodies. In another embodiment, the nucleic acidis isolated from bacteria or phage.

In some embodiments, a nucleic acid encoding a heavy chain of ananti-ErbB2 antibody of the invention can comprise a nucleotide sequenceencoding a V_(H) domain of the invention joined in-frame to a nucleotidesequence encoding a heavy chain constant domain from any source.Similarly, a nucleic acid molecule encoding a light chain of ananti-ErbB2 antibody of the invention can comprise a nucleotide sequenceencoding a V_(L) domain of the invention joined in-frame to a nucleotidesequence encoding a light chain constant domain from any source.

In a further aspect of the invention, nucleic acid molecules encodingthe variable domain of the heavy (V_(H)) and/or light (V_(L)) chains are“converted” to full-length antibody genes. In one embodiment, nucleicacid molecules encoding the V_(H) or V_(L) domains are converted tofull-length antibody genes by insertion into an expression vectoralready encoding heavy chain constant (C_(H)) or light chain constant(C_(L)) domains, respectively, such that the V_(H) segment isoperatively linked to the C_(H) segment(s) within the vector, and/or theV_(L) segment is operatively linked to the C_(L) segment within thevector. In another embodiment, nucleic acid molecules encoding the V_(H)and/or V_(L) domains are converted into full-length antibody genes bylinking, e.g., ligating, a nucleic acid molecule encoding a V_(H) and/orV_(L) domains to a nucleic acid molecule encoding a C_(H) and/or C_(L)domain using standard molecular biological techniques. Nucleotidesequences of human heavy and light chain immunoglobulin constant domaingenes are known in the art. See, e.g., Kabat et al. Sequences ofProteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242,1991. Nucleic acid molecules encoding the full-length heavy and/or lightchains may then be expressed from a cell into which they have beenintroduced and the anti-ErbB2 antibody isolated.

The nucleic acid molecules may be used to recombinantly express largequantities of anti-ErbB2 antibodies. The nucleic acid molecules also maybe used to produce chimeric antibodies, bispecific antibodies, singlechain antibodies, immunoadhesins, diabodies, mutated antibodies andantibody derivatives, as described further below. If the nucleic acidmolecules are derived from a non-human, non-transgenic animal, thenucleic acid molecules may be used for antibody humanization, also asdescribed below.

In another embodiment, a nucleic acid molecule of the invention is usedas a probe or PCR primer for a specific antibody sequence. For instance,the nucleic acid can be used as a probe in diagnostic methods or as aPCR primer to amplify regions of DNA that could be used, inter alia, toisolate additional nucleic acid molecules encoding variable domains ofanti-ErbB2 antibodies. In some embodiments, the nucleic acid moleculesare oligonucleotides. In some embodiments, the oligonucleotides are fromhighly variable domains of the heavy and light chains of the antibody ofinterest. In some embodiments, the oligonucleotides encode all or a partof one or more of the CDRs of antibodies 1.44.1, 1.140, 1.43, 1.14.1,1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3 (or variants thereofas described herein).

Vectors

The invention provides vectors comprising nucleic acid molecules thatencode the heavy chain of an anti-ErbB2 antibody of the invention or anantigen-binding portion thereof, nucleic acid molecules that encode thelight chain of such antibodies or antigen-binding portion thereof, orboth or a targed binding agent. The invention further provides vectorscomprising nucleic acid molecules encoding fusion proteins, modifiedantibodies, antibody fragments, and probes thereof.

In some embodiments, the anti-ErbB2 antibodies or antigen-bindingportions of the invention are expressed by inserting DNAs encodingpartial or full-length light and/or heavy chains, obtained as describedabove, into expression vectors such that the genes are operativelylinked to necessary expression control sequences such as transcriptionaland translational control sequences. Expression vectors includeplasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV),plant viruses such as cauliflower mosaic virus, tobacco mosaic virus,cosmids, YACs, EBV derived episomes, and the like. The antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevectors. In some embodiments, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can easily beinserted and expressed, as described above. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C domain, and also atthe splice regions that occur within the human C_(H) exons.Polyadenylation and transcription termination occur at nativechromosomal sites downstream of the coding regions. The recombinantexpression vector also can encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The antibody chaingene may be cloned into the vector such that the signal peptide islinked in-frame to the amino terminus of the immunoglobulin chain. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. For further description ofviral regulatory elements, and sequences thereof, see e.g., U.S. Pat.No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615.Methods for expressing antibodies in plants, including a description ofpromoters and vectors, as well as transformation of plants is known inthe art. See, e.g., U.S. Pat. No. 6,517,529, incorporated herein byreference. Methods of expressing polypeptides in bacterial cells orfungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, incorporated herein by reference).For example, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification), the neo gene (for G418selection), and the glutamate synthetase gene.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding targed binding agent and/or anti-ErbB2antibodies and vectors comprising these nucleic acid molecules can beused for transfection of a suitable mammalian, plant, bacterial or yeasthost cell. Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, e.g., U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporatedherein by reference). Methods of transforming plant cells are well knownin the art, including, e.g., Agrobacterium-mediated transformation,biolistic transformation, direct injection, electroporation and viraltransformation. Methods of transforming bacterial and yeast cells arealso well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, N50 cells, SP2 cells, HEK-293T cells,NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, Africangreen monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, and a number of other cell lines. Cell linesof particular preference are selected through determining which celllines have high expression levels. Other cell lines that may be used areinsect cell lines, such as SIP or Sf21 cells. When recombinantexpression vectors encoding antibody genes are introduced into mammalianhost cells, the antibodies are produced by culturing the host cells fora period of time sufficient to allow for expression of the antibody inthe host cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods. Plant host cells include, e.g., Nicotiana, Arabidopsis,duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coliand Streptomyces species. Yeast host cells include Schizosaccharomycespombe, Saccharomyces cerevisiae and Pichia pastoris.

Further, expression of antibodies of the invention from production celllines can be enhanced using a number of known techniques. For example,the glutamine synthetase gene expression system (the GS system) is acommon approach for enhancing expression under certain conditions. TheGS system is discussed in whole or part in connection with EuropeanPatent Nos. 0 216 846, 0 256 055, 0 323 997 and 0 338 841.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant invention, regardless of the glycosylation of theantibodies.

Transgenic Animals and Plants

Anti-ErbB2 antibodies of the invention also can be producedtransgenically through the generation of a mammal or plant that istransgenic for the immunoglobulin heavy and light chain sequences ofinterest and production of the antibody in a recoverable form therefrom.In connection with the transgenic production in mammals, anti-ErbB2antibodies can be produced in, and recovered from, the milk of goats,cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687,5,750,172, and 5,741,957, incorporated herein by reference. In someembodiments, non-human transgenic animals that comprise humanimmunoglobulin loci are immunized with ErbB2 or an immunogenic portionthereof, as described above. Methods for making antibodies in plants aredescribed, e.g., in U.S. Pat. Nos. 6,046,037 and 5,959,177, incorporatedherein by reference.

In some embodiments, non-human transgenic animals or plants are producedby introducing one or more nucleic acid molecules encoding an anti-ErbB2antibody of the invention into the animal or plant by standardtransgenic techniques. See Hogan and U.S. Pat. No. 6,417,429, supra. Thetransgenic cells used for making the transgenic animal can be embryonicstem cells or somatic cells or a fertilized egg. The transgenicnon-human organisms can be chimeric, nonchimeric heterozygotes, andnonchimeric homozygotes. See, e.g., Hogan et al., Manipulating the MouseEmbryo: A Laboratory Manual 2^(nd) ed., Cold Spring Harbor Press (1999);Jackson et al., Mouse Genetics and Transgenics: A Practical Approach,Oxford University Press (2000); and Pinkert, Transgenic AnimalTechnology: A Laboratory Handbook, Academic Press (1999), allincorporated herein by reference. In some embodiments, the transgenicnon-human animals have a targeted disruption and replacement by atargeting construct that encodes a heavy chain and/or a light chain ofinterest. In another embodiment, the transgenic animals comprise andexpress nucleic acid molecules encoding heavy and light chains thatspecifically bind to ErbB2, preferably human ErbB2. In some embodiments,the transgenic animals comprise nucleic acid molecules encoding amodified antibody such as a single-chain antibody, a chimeric antibodyor a humanized antibody. The anti-ErbB2 antibodies may be made in anytransgenic animal. In another embodiment, the non-human animals aremice, rats, sheep, pigs, goats, cattle or horses. The non-humantransgenic animal expresses said encoded polypeptides in blood, milk,urine, saliva, tears, mucus and other bodily fluids.

Phage Display Libraries

The invention provides a method for producing an anti-ErbB2 antibody oran antigen-binding portion thereof comprising the steps of synthesizinga library of human antibodies on phage, screening the library with ErbB2or a portion thereof, isolating phage that bind ErbB2, and obtaining theantibody from the phage. By way of example, one method for preparing thelibrary of antibodies for use in phage display techniques comprises thesteps of immunizing a non-human animal comprising human immunoglobulinloci with ErbB2 or an antigenic portion thereof to create an immuneresponse, extracting antibody-producing cells from the immunized animal;isolating RNA encoding heavy and light chains of antibodies of theinvention from the extracted cells, reverse transcribing the RNA toproduce cDNA, amplifying the cDNA using primers, and inserting the cDNAinto a phage display vector such that antibodies are expressed on thephage. Recombinant anti-ErbB2 antibodies of the invention may beobtained in this way.

Recombinant anti-ErbB2 human antibodies of the invention can be isolatedby screening a recombinant combinatorial antibody library. Preferablythe library is a scFv phage display library, generated using human V_(L)and V_(H) cDNAs prepared from mRNA isolated from B cells. Methods forpreparing and screening such libraries are known in the art. Kits forgenerating phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612). Therealso are other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791,WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); McCafferty etal., Nature 348:552-554 (1990); Griffiths et al., EMBO J. 12:725-734(1993); Hawkins et al., J. Mol. Biol. 226:889-896 (1992); Clackson etal., Nature 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA89:3576-3580 (1992); Garrad et al., Bio/Technology 9:1373-1377 (1991);Hoogenboom et al., Nuc. Acid Res. 19:4133-4137 (1991); and Barbas etal., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991), all incorporatedherein by reference.

In one embodiment, to isolate and produce human anti-ErbB2 antibodieswith the desired characteristics, a human anti-ErbB2 antibody asdescribed herein is first used to select human heavy and light chainsequences having similar binding activity toward ErbB2, using theepitope imprinting methods described in PCT Publication No. WO 93/06213,incorporated herein by reference. The antibody libraries used in thismethod are preferably scFv libraries prepared and screened as describedin PCT Publication No. WO 92/01047, McCafferty et al., Nature348:552-554 (1990); and Griffiths et al., EMBO J. 12:725-734 (1993), allincorporated herein by reference. The scFv antibody libraries preferablyare screened using human ErbB2 as the antigen.

Once initial human V_(L) and V_(H) domains are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for ErbB2 binding toselect preferred V_(L)/V_(H) pair combinations. Additionally, to furtherimprove the quality of the antibody, the V_(L) and V_(H) segments of thepreferred V_(L)/V_(H) pair(s) can be randomly mutated, preferably withinthe CDR3 region of V_(H) and/or V_(L), in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) domainsusing PCR primers complimentary to the V_(H) CDR3 or V_(L) CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be re-screened for bindingto ErbB2.

Following screening and isolation of an anti-ErbB2 antibody of theinvention from a recombinant immunoglobulin display library, nucleicacids encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can further be manipulated to create other antibodyforms of the invention, as described below. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described above.

Class Switching

Another aspect of the invention provides a method for converting theclass or subclass of an anti-ErbB2 antibody to another class orsubclass. In some embodiments, a nucleic acid molecule encoding a V_(L)or V_(H) that does not include sequences encoding C_(L) or C_(H) isisolated using methods well-known in the art. The nucleic acid moleculethen is operatively linked to a nucleotide sequence encoding a C_(L) orC_(H) from a desired immunoglobulin class or subclass. This can beachieved using a vector or nucleic acid molecule that comprises a C_(L)or C_(H) chain, as described above. For example, an anti-ErbB2 antibodythat was originally IgM can be class switched to an IgG. Further, theclass switching may be used to convert one IgG subclass to another,e.g., from IgG1 to IgG2. Another method for producing an antibody of theinvention comprising a desired isotype comprises the steps of isolatinga nucleic acid encoding a heavy chain of an anti-ErbB2 antibody and anucleic acid encoding a light chain of an anti-ErbB2 antibody, isolatingthe sequence encoding the V_(H) region, ligating the V_(H) sequence to asequence encoding a heavy chain constant domain of the desired isotype,expressing the light chain gene and the heavy chain construct in a cell,and collecting the anti-ErbB2 antibody with the desired isotype.

Deimmunized Antibodies

In another aspect of the invention, the antibody may be deimmunized toreduce its immunogenicity using the techniques described in, e.g., PCTPublication Nos. WO98/52976 and WO00/34317 (incorporated herein byreference).

Mutated Antibodies

In another embodiment, the nucleic acid molecules, vectors and hostcells may be used to make mutated anti-ErbB2 antibodies. The antibodiesmay be mutated in the variable domains of the heavy and/or light chains,e.g., to alter a binding property of the antibody. For example, amutation may be made in one or more of the CDR regions to increase ordecrease the K_(D) of the antibody for ErbB2, to increase or decreasek_(off), or to alter the binding specificity of the antibody. Techniquesin site-directed mutagenesis are well-known in the art. See, e.g.,Sambrook et al. and Ausubel et al., supra. In another embodiment, one ormore mutations are made at an amino acid residue that is known to bechanged compared to the germline in antibody 1.44.1, 1.140, 1.43,1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and 1.71.3. Themutations may be made in a CDR region or framework region of a variabledomain, or in a constant domain. In another embodiment, the mutationsare made in a variable domain. In some embodiments, one or moremutations are made at amino acid residues that are known to be changedcompared to the germline in a CDR region or framework region of avariable domain of an amino acid sequence selected from SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, and 44, or whose nucleotide sequence is presented in SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, or 43.

In another embodiment, the framework region is mutated so that theresulting framework region(s) have the amino acid sequence of thecorresponding germline gene. A mutation may be made in a frameworkregion or constant domain to increase the half-life of the anti-ErbB2antibody. See, e.g., PCT Publication No. WO 00/09560, incorporatedherein by reference. A mutation in a framework region or constant domainalso can be made to alter the immunogenicity of the antibody, to providea site for covalent or non-covalent binding to another molecule, or toalter such properties as complement fixation, FcR binding andantibody-dependent cell-mediated cytotoxicity (ADCC). According to theinvention, a single antibody may have mutations in any one or more ofthe CDRs or framework regions of the variable domain or in the constantdomain.

In some embodiments, there are from 1 to 8, including any number inbetween, amino acid mutations in either the V_(H) or V_(L) domains ofthe mutated anti-ErbB2 antibody compared to the anti-ErbB2 antibodyprior to mutation. In any of the above, the mutations may occur in oneor more CDR regions. Further, any of the mutations can be conservativeamino acid substitutions. In some embodiments, there are no more than 5,4, 3, 2, or 1 amino acid changes in the constant domains.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be madethat comprises all or a portion of an anti-ErbB2 antibody of theinvention linked to another polypeptide. In another embodiment, only thevariable domains of the anti-ErbB2 antibody are linked to thepolypeptide. In another preferred embodiment, the V_(H) domain of ananti-ErbB2 antibody is linked to a first polypeptide, while the V_(L)domain of an anti-ErbB2 antibody is linked to a second polypeptide thatassociates with the first polypeptide in a manner such that the V_(H)and V_(L) domains can interact with one another to form an antigenbinding site. In another preferred embodiment, the V_(H) domain isseparated from the V_(L) domain by a linker such that the V_(H) andV_(L) domains can interact with one another (see below under SingleChain Antibodies). The V_(H)-linker-V_(L) antibody is then linked to thepolypeptide of interest. The fusion antibody is useful for directing apolypeptide to an ErbB2-expressing cell or tissue. The polypeptide maybe a therapeutic agent, such as a toxin, growth factor or otherregulatory protein, or may be a diagnostic agent, such as an enzyme thatmay be easily visualized, such as horseradish peroxidase. In addition,fusion antibodies can be created in which two (or more) single-chainantibodies are linked to one another. This is useful if one wants tocreate a divalent or polyvalent antibody on a single polypeptide chain,or if one wants to create a bispecific antibody.

To create a single chain antibody, (scFv) the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃,such that the V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) domains joined by theflexible linker. See, e.g., Bird et al., Science 242:423-426 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);McCafferty et al., Nature 348:552-554 (1990). The single chain antibodymay be monovalent, if only a single V_(H) and V_(L) are used, bivalent,if two V_(H) and V_(L) are used, or polyvalent, if more than two V_(H)and V_(L) are used. Bispecific or polyvalent antibodies may be generatedthat bind specifically to ErbB2 and to another molecule.

In other embodiments, other modified antibodies may be prepared usinganti-ErbB2 antibody encoding nucleic acid molecules. For instance,“Kappa bodies” (Ill et al., Protein. Eng. 10: 949-57 (1997)),“Minibodies” (Martin et al., EMBO J. 13: 5303-9 (1994)), “Diabodies”(Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or“Janusins” (Traunecker et al., EMBO J. 10:3655-3659 (1991) andTraunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) may beprepared using standard molecular biological techniques following theteachings of the specification.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of ErbB2. In some embodiments, the bispecificantibody has a first heavy chain and a first light chain from antibody1.44.1, 1.140, 1.43, 1.14.1, 1.100.1, 1.96, 1.18.1, 1.20, 1.39, 1.24 and1.71.3 and an additional antibody heavy chain and light chain. In someembodiments, the additional light chain and heavy chain also are fromone of the above-identified monoclonal antibodies, but are differentfrom the first heavy and light chains.

In some embodiments, the modified antibodies described above areprepared using one or more of the variable domains or CDR regions from ahuman anti-ErbB2 monoclonal antibody provided herein.

The antibodies of the invention also encompass antibodies that havehalf-lives (e.g., serum half-lives) in a mammal, preferably a human, ofgreater than that of an unmodified antibody. In one embodiment, saidantibody half life is greater than about 15 days, greater than about 20days, greater than about 25 days, greater than about 30 days, greaterthan about 35 days, greater than about 40 days, greater than about 45days, greater than about 2 months, greater than about 3 months, greaterthan about 4 months, or greater than about 5 months. The increasedhalf-lives of the antibodies of the present invention or fragmentsthereof in a mammal, preferably a human, result in a higher serum titerof said antibodies or antibody fragments in the mammal, and thus, reducethe frequency of the administration of said antibodies or antibodyfragments and/or reduces the concentration of said antibodies orantibody fragments to be administered. Antibodies or fragments thereofhaving increased in vivo half-lives can be generated by techniques knownto those of skill in the art. For example, antibodies or fragmentsthereof with increased in vivo half-lives can be generated by modifying(e.g., substituting, deleting or adding) amino acid residues identifiedas involved in the interaction between the Fc domain and the FcRnreceptor (see, e.g., International Publication Nos. WO 97/34631 and WO02/060919, which are incorporated herein by reference in theirentireties). Antibodies or fragments thereof with increased in vivohalf-lives can be generated by attaching to said antibodies or antibodyfragments polymer molecules such as high molecular weightpolyethyleneglycol (PEG). PEG can be attached to said antibodies orantibody fragments with or without a multifunctional linker eitherthrough site-specific conjugation of the PEG to the N- or C-terminus ofsaid antibodies or antibody fragments or via epsilon-amino groupspresent on lysine residues. Linear or branched polymer derivatizationthat results in minimal loss of biological activity will be used. Thedegree of conjugation will be closely monitored by SDS-PAGE and massspectrometry to ensure proper conjugation of PEG molecules to theantibodies. Unreacted PEG can be separated from antibody-PEG conjugatesby, e.g., size exclusion or ion-exchange chromatography.

An antigen binding site may be provided by means of arrangement of CDRson non-antibody protein scaffolds, such as fibronectin or cytochrome Betc. (Haan & Maggos (2004) BioCentury, 12(5): A1-A6; Koide et al. (1998)Journal of Molecular Biology, 284: 1141-1151; Nygren et al. (1997)Current Opinion in Structural Biology, 7: 463-469) or by randomising ormutating amino acid residues of a loop within a protein scaffold toconfer binding specificity for a desired target. Scaffolds forengineering novel binding sites in proteins have been reviewed in detailby Nygren et al. (Nygren et al. (1997) Current Opinion in StructuralBiology, 7: 463-469). Protein scaffolds for antibody mimics aredisclosed in WO/0034784, which is herein incorporated by reference inits entirety, in which the inventors describe proteins (antibody mimics)that include a fibronectin type III domain having at least onerandomised loop. A suitable scaffold into which to graft one or moreCDRs, e.g. a set of HCDRs, may be provided by any domain member of theimmunoglobulin gene superfamily. The scaffold may be a human ornon-human protein. An advantage of a non-antibody protein scaffold isthat it may provide an antigen-binding site in a scaffold molecule thatis smaller and/or easier to manufacture than at least some antibodymolecules. Small size of a binding member may confer usefulphysiological properties, such as an ability to enter cells, penetratedeep into tissues or reach targets within other structures, or to bindwithin protein cavities of the target antigen. Use of antigen bindingsites in non-antibody protein scaffolds is reviewed in Wess, 2004 (Wess,L. In: BioCentury, The Bernstein Report on BioBusiness, 12(42), A1-A7,2004). Typical are proteins having a stable backbone and one or morevariable loops, in which the amino acid sequence of the loop or loops isspecifically or randomly mutated to create an antigen-binding site thatbinds the target antigen. Such proteins include the IgG-binding domainsof protein A from S. aureus, transferrin, albumin, tetranectin,fibronectin (e.g. 10th fibronectin type III domain), lipocalins as wellas gamma-crystalline and other Affilin™ scaffolds (Scil Proteins).Examples of other approaches include synthetic “Microbodies” based oncyclotides—small proteins having intra-molecular disulphide bonds,Microproteins (Versabodies™, Amunix) and ankyrin repeat proteins(DARPins, Molecular Partners).

In addition to antibody sequences and/or an antigen-binding site, atargeted binding agent according to the present invention may compriseother amino acids, e.g. forming a peptide or polypeptide, such as afolded domain, or to impart to the molecule another functionalcharacteristic in addition to ability to bind antigen. Targeted bindingagents of the invention may carry a detectable label, or may beconjugated to a toxin or a targeting moiety or enzyme (e.g. via apeptidyl bond or linker). For example, a targeted binding agent maycomprise a catalytic site (e.g. in an enzyme domain) as well as anantigen binding site, wherein the antigen binding site binds to theantigen and thus targets the catalytic site to the antigen. Thecatalytic site may inhibit biological function of the antigen, e.g. bycleavage.

Derivatized and Labeled Antibodies

An anti-ErbB2 antibody or antigen-binding portion of the invention canbe derivatized or linked to another molecule (e.g., another peptide orprotein). In general, the antibodies or portion thereof are derivatizedsuch that the ErbB2 binding is not affected adversely by thederivatization or labeling. Accordingly, the antibodies and antibodyportions of the invention are intended to include both intact andmodified forms of the human anti-ErbB2 antibodies described herein. Forexample, an antibody or antibody portion of the invention can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (e.g., a bispecific antibody or a diabody), adetection agent, a cytotoxic agent, a pharmaceutical agent, and/or aprotein or peptide that can mediate association of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Il.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antigen-binding portion ofthe invention may be derivatized include fluorescent compounds,including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody can also be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. When an antibody is labeled with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce areaction product that can be discerned. For example, when the agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody can also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody can also be labeled with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

An anti-ErbB2 antibody can also be labeled with a radiolabeled aminoacid. The radiolabel can be used for both diagnostic and therapeuticpurposes. For instance, the radiolabel can be used to detectErbB2-expressing cells by x-ray or other diagnostic techniques. Further,the radiolabel can be used therapeutically as a toxin forErbB2-expressing cells, such as those which cause unwanted immuneresponse. Examples of labels for polypeptides include, but are notlimited to, the following radioisotopes or radionuclides: ³H, ¹⁴C, ¹⁵N,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I and ¹³¹I.

In some embodiments, the anti-ErbB2 antibody can be labeled with aparamagnetic, radioactive or fluorogenic ion that is detectable uponimaging. In some embodiments, the paramagnetic ion is chromium (III),manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper(II), neodymium (III), samarium (III), ytterbium (III), gadolinium(III), vanadium (II), terbium (III), dysprosium (III), holmium (III) orerbium (III). In other embodiments, the radioactive ion is iodine123,technetium99, indium111, rhenium188, rhenium186, copper67, iodine131,yttrium90, iodine125, astatine211, and gallium67. In other embodiments,the anti-ErbB2 antibody is labeled with an X-ray imaging agent such aslanthanum (III), gold (III) lead (II) and bismuth (III). An anti-ErbB2antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups are useful to improve the biological characteristicsof the antibody, e.g., to increase serum half-life or to increase tissuebinding.

Pharmaceutical Compositions and Kits

The invention relates to compositions comprising a targed binding agentand/or human anti-ErbB2 antibody with antagonist properties for thetreatment of subjects in need of a therapeutic procedure including, butnot limited to, those afflicted with cancer. In some embodiments, thesubject of treatment is a human. In other embodiments, the subject is aveterinary subject.

Treatment may involve administration of one or more inhibitoryanti-ErbB2 monoclonal antibodies of the invention, or antigen-bindingfragments thereof, alone or with a pharmaceutically acceptable carrier.Inhibitory anti-ErbB2 antibodies of the invention and compositionscomprising them, can be administered in combination with one or moreother therapeutic, diagnostic or prophylactic agents.

In certain embodiments, the therapeutic agents of the disclosure mayinclude antineoplastic agents. Antineoplastic agents include, withoutlimitation, platinum-based agents, such as carboplatin and cisplatin;nitrogen mustard alkylating agents; nitrosourea alkylating agents, suchas carmustine (BCNU) and other alkylating agents; antimetabolites, suchas methotrexate; purine analog antimetabolites; pyrimidine analogantimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonalantineoplastics, such as goserelin, leuprolide, and tamoxifen; naturalantineoplastics, such as taxanes (e.g., docetaxel and paclitaxel),aldesleukin, interleukin-2, etoposide (VP-16), interferon alpha, andtretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin,dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vincaalkaloid natural antineoplastics, such as vinblastine and vincristine.

In various embodiments, the antineoplastic agent is 5-Fluoruracil,6-mercatopurine, Actinomycin, Adriamycin®, Admen®, Aminoglutethimide,Anastrozole, Aredia®, Arimidex®, Aromasin®, Bonefos®, Bleomycin,carboplatin, Cactinomycin, Capecitabine, Cisplatin, Clodronate,Cyclophosphamide, Cytadren®, Cytoxan®, Dactinomycin, Docetaxel, Doxyl®,Doxorubicin, Epirubicin, Etoposide, Exemestane, Femora®, Fluorouracil,Fluoxymesterone, Halotestin®, Herceptin®®, Letrozole, Leucovorincalcium, Megace®, Megestrol acetate, Methotrexate, Mitomycin,Mitoxantrone, Mutamycin®, Navelbine®, Nolvadex®, Novantrone®, Oncovin®,Ostac®, Paclitaxel, Pamidronate, Pharmorubicin®, Platinol®, prednisone,Procytox®, Tamofen®, Tamone®, Tamoplex®, Tamoxifen, Taxol®, Taxotere®,Trastuzumab, Thiotepa, Velbe®, Vepesid®, Vinblastine, Vincristine,Vinorelbine, Xeloda®, or a combination thereof.

In some embodiments, the antineoplastic agent comprises a monoclonalantibody, a humanized antibody, a chimeric antibody, a single chainantibody, or a fragment of an antibody. Exemplary antibodies include,but are not limited to, Rituxan, IDEC-C2B8, anti-CD20 Mab, Panorex,3622W94, anti-EGP40 (17-1A) pancarcinoma antigen on adenocarcinomasHerceptin®, Erbitux, anti-Her2, Anti-EGFr, BEC2, anti-idiotypic-GD₃epitope, Ovarex, B43.13, anti-idiotypic CA125, 4B5, Anti-VEGF, RhuMAb,MDX-210, anti-HER2, MDX-22, MDX-220, MDX-447, MDX-260, anti-GD-2,Quadramet, CYT-424, IDEC-Y2B8, Oncolym, Lym-1, SMART M195, ATRAGEN,LDP-03, anti-CAMPATH, for t6, anti CD6, MDX-11, OV103, Zenapax,Anti-Tac, anti-IL-2 receptor, MELIMMUNE-2, MELIMM UN E-1, CEACIDE,Pretarget, NovoMAb-G2, TNT, anti-histone, Gliomab-H, GNI-250, EMD-72000,LymphoCide, CMA 676, Monopharm-C, anti-FLK-2, SMART 1D10, SMART ABL 364,ImmuRAIT-CEA, or combinations thereof.

In yet another embodiment, the antineoplastic agent comprises anadditional type of tumor cell. In a specific embodiment, the additionaltype of tumor cell is a MCF-10A, MCF-10F, MCF-10-2A, MCF-12A, MCF-12F,ZR-75-1, ZR-75-30, UACC-812, UACC-893, HCC38, HCC70, HCC202, HCC1007 BL,HCC1008, HCC1143, HCC1187, HCC1187 BL, HCC1395, HCC1569, HCC1599,HCC1599 BL, HCC1806, HCC1937, HCC1937 BL, HCC1954, HCC1954 BL, HCC2157,Hs 274.T, Hs 281.T, Hs 343.T, Hs 362.T, Hs 574.T, Hs 579.Mg, Hs 605.T,Hs 742.T, Hs 748.T, Hs 875.T, MB 157, SW527, 184A1, 184B5, MDA-MB-330,MDA-MB-415, MDA-MB-435S, MDA-MB-436, MDA-MB-453, MDA-MB-468 RT4, BT-474,CAMA-1, MCF7 [MCF-7], MDA-MB-134-VI, MDA-MB-157, MDA-MB-175-VII HTB-27MDA-MB-361, SK-BR-3 or ME-180 cell, all of which are available fromATTC.

In still further embodiments, the antineoplastic agent comprises anantisense reagent, such as an siRNA or a hairpin RNA molecule, whichreduces the expression or function of a gene that is expressed in acancer cell. Exemplary antisense reagents which may be used includethose directed to mucin, Ha-ras, VEGFR1 or BRCA1. Such reagents aredescribed in U.S. Pat. No. 6,716,627 (mucin), U.S. Pat. No. 6,723,706(Ha-ras), U.S. Pat. No. 6,710,174 (VEGFR1) and in U.S. PatentPublication No. 2004/0014051 (BRCA1).

In further embodiments, the antineoplastic agent comprises cellsautologous to the subject, such as cells of the immune system such asmacrophages, T cells or dendrites. In some embodiments, the cells havebeen treated with an antigen, such as a peptide or a cancer antigen, orhave been incubated with tumor cells from the patient. In oneembodiment, autologous peripheral blood lymphocytes may be mixed withSV-BR-1 cells and administered to the subject. Such lymphocytes may beisolated by leukaphoresis. Suitable autologous cells which may be used,methods for their isolation, methods of modifying said cells to improvetheir effectiveness and formulations comprising said cells are describedin U.S. Pat. Nos. 6,277,368, 6,451,316, 5,843,435, 5,928,639, 6,368,593and 6,207,147, and in International PCT Publications Nos. WO04/021995and WO00/57705.

In some embodiments of the methods described herein directed to thetreatment of cancer, the subject is treated prior to, concurrently with,or subsequently to the treatment with the cells of the presentinvention, with a complementary therapy to the cancer, such as surgery,chemotherapy, radiation therapy, or hormonal therapy or a combinationthereof.

In a specific embodiment where the cancer is breast cancer, thecomplementary treatment may comprise breast-sparing surgery i.e. anoperation to remove the cancer but not the breast, also calledbreast-sparing surgery, breast-conserving surgery, lumpectomy, segmentalmastectomy, or partial mastectomy. In another embodiment, it comprises amastectomy. A mastectomy is an operation to remove the breast, or asmuch of the breast tissue as possible, and in some cases also the lymphnodes under the arm. In yet another embodiment, the surgery comprisessentinel lymph node biopsy, where only one or a few lymph nodes (thesentinel nodes) are removed instead of removing a much larger number ofunderarm lymph nodes. Surgery may also comprise modified radicalmastectomy, where a surgeon removes the whole breast, most or all of thelymph nodes under the arm, and, often, the lining over the chestmuscles. The smaller of the two chest muscles also may be taken out tomake it easier to remove the lymph nodes.

In a specific embodiment, the complementary treatment comprisesradiation therapy. Radiation therapy may comprise external radiation,where radiation comes from a machine, or from internal radiation(implant radiation, wherein the radiation originates from radioactivematerial placed in thin plastic tubes put directly in the breast.

In another specific embodiment, the complementary treatment compriseschemotherapy. Chemotherapeutic agents found to be of assistance in thesuppression of tumors include but are not limited to alkylating agents(e.g., nitrogen mustards), antimetabolites (e.g., pyrimidine analogs),radioactive isotopes (e.g., phosphorous and iodine), miscellaneousagents (e.g., substituted ureas) and natural products (e.g., vincaalkyloids and antibiotics). In a specific embodiment, thechemotherapeutic agent is selected from the group consisting ofallopurinol sodium, dolasetron mesylate, pamidronate disodium,etidronate, fluconazole, epoetin alfa, levamisole HCL, amifostine,granisetron HCL, leucovorin calcium, sargramostim, dronabinol, mesna,filgrastim, pilocarpine HCL, octreotide acetate, dexrazoxane,ondansetron HCL, ondansetron, busulfan, carboplatin, cisplatin,thiotepa, melphalan HCL, melphalan, cyclophosphamide, ifosfamide,chlorambucil, mechlorethamine HCL, carmustine, lomustine, polifeprosan20 with carmustine implant, streptozocin, doxorubicin HCL, bleomycinsulfate, daunirubicin HCL, dactinomycin, daunoruebicin citrate,idarubicin HCL, plimycin, mitomycin, pentostatin, mitoxantrone,valrubicin, cytarabine, fludarabine phosphate, floxuridine, cladribine,methotrexate, mercaptipurine, thioguanine, capecitabine,methyltestosterone, nilutamide, testolactone, bicalutamide, flutamide,anastrozole, toremifene citrate, estramustine phosphate sodium, ethinylestradiol, estradiol, esterified estrogens, conjugated estrogens,leuprolide acetate, goserelin acetate, medroxyprogesterone acetate,megestrol acetate, levamisole HCL, aldesleukin, irinotecan HCL,dacarbazine, asparaginase, etoposide phosphate, gemcitabine HCL,altretamine, topotecan HCL, hydroxyurea, interferon alfa-2b, mitotane,procarbazine HCL, vinorelbine tartrate, E. coli L-asparaginase, ErwiniaL-asparaginase, vincristine sulfate, denileukin diftitox, aldesleukin,rituximab, interferon alfa-2a, paclitaxel, docetaxel, BCG live(intravesical), vinblastine sulfate, etoposide, tretinoin, teniposide,porfimer sodium, fluorouracil, betamethasone sodium phosphate andbetamethasone acetate, letrozole, etoposide citrororum factor, folinicacid, calcium leucouorin, 5-fluorouricil, adriamycin, cytoxan, anddiamino dichloro platinum, said chemotherapy agent in combination withthymosinα₁ being administered in an amount effective to reduce said sideeffects of chemotherapy in said patient.

In another specific embodiment, the complementary treatment compriseshormonal therapy. Hormonal therapy may comprise the use of a drug, suchas tamoxifen, that can block the natural hormones like estrogen or maycomprise aromatase inhibitors which prevent the synthesis of estradiol.Alternative, hormonal therapy may comprise the removal of the subject'sovaries, especially if the subject is a woman who has not yet gonethrough menopause.

As used herein, “pharmaceutically acceptable carrier” means any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Some examples of pharmaceutically acceptablecarriers are water, saline, phosphate buffered saline, dextrose,glycerol, ethanol and the like, as well as combinations thereof. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Additional examples of pharmaceutically acceptablesubstances are wetting agents or minor amounts of auxiliary substancessuch as wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the antibody.

The compositions of this invention may be in a variety of forms, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Thepreferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans. The preferred mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In another embodiment, the antibody isadministered by intravenous infusion or injection. In another preferredembodiment, the antibody is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the anti-ErbB2 antibody inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The antibodies of the present invention can be administered by a varietyof methods known in the art, although for many therapeutic applications,the preferred route/mode of administration is subcutaneous,intramuscular, or intravenous infusion. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results.

In certain embodiments, the antibody compositions active compound may beprepared with a carrier that will protect the antibody against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid, chitosan and alginate. Many methodsfor the preparation of such formulations are patented or generally knownto those skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978).

The invention also provides compositions suitable for administration byinhalation, which comprise the anti-ErbB2 antibodies described herein.The anti-ErbB2 antibodies may be conveniently delivered to a subject inthe form of an aerosol spray presentation from pressurized packs or froma nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. Dellamary et al., (2004) J Control Release.;95(3): 489-500 describes formulations for the pulmonary delivery ofantibodies.

The invention also provides compositions, suitable for administrationthrough the oral mucosa, which comprise the anti-ErbB2 antibodydescribed herein. Oral transmucosal delivery refers to the delivery of adelivery vehicle across a mucous membrane in the oral cavity, pharyngealcavity, or esophagus, and may be contrasted, for example, withtraditional oral delivery, in which absorption of a drug occurs in theintestine. Accordingly, routes of administration in which the anti-ErbB2antibodies are absorbed through the buccal, sublingual, gingival,pharyngeal, and/or esophageal mucosa are all encompassed within “oraltransmucosal delivery,” as that term is used herein. For administrationthrough the transmucosal mucosa, the anti-ErbB2 antibody may beformulated, for example, into chewing gums (see U.S. Pat. No. 5,711,961)or buccal patches (see e.g. U.S. Pat. No. 5,298,256).

The invention also provides compositions suitable for administrationthrough the vaginal mucosa, which comprise the anti-ErbB2 antibodiesdescribed herein. The anti-ErbB2 antibodies of the invention may beformulated into a vaginal suppository, foam, cream, tablet, capsule,ointment, or gel.

In certain embodiments, the pharmaceutical compositions comprising theanti-ErbB2 antibodies are formulated with permeants appropriate to thetransmucosal barrier to be permeated. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives.

In certain embodiments, an anti-ErbB2 antibody of the invention isorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) can also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the anti-ErbB2 antibodies canbe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

Additional active compounds also can be incorporated into thecompositions. In certain embodiments, an inhibitory anti-ErbB2 antibodyof the invention is co-formulated with and/or co-administered with oneor more additional therapeutic agents, such as those listed supra. Suchcombination therapies may require lower dosages of the inhibitoryanti-ErbB2 antibody as well as the co-administered agents, thus avoidingpossible toxicities or complications associated with the variousmonotherapies.

Inhibitory anti-ErbB2 antibodies of the invention and compositionscomprising them also may be administered in combination with othertherapeutic regimens, in particular in combination with surgicalradiological and/or chemotherapy treatment.

The compositions of the invention may include a “therapeuticallyeffective amount” or a “prophylactically effective amount” of anantibody or antigen-binding portion of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antibody portion mayvary according to factors such as the disease state, age, sex, andweight of the subject, and the ability of the antibody or antibodyportion to elicit a desired response in the subject. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the antibody or antibody portion are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount may be less thanthe therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the close can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the anti-ErbB2 antibody or portion thereof and theparticular therapeutic or prophylactic effect to be achieved, and (b)the limitations inherent in the art of compounding such an antibody forthe treatment of sensitivity in subjects.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg,more preferably 0.1 to 25, 0.1 to 10 or 0.1 to 3 mg/kg. In someembodiments, a formulation contains 5 mg/ml of antibody in a buffer of20 mM sodium citrate, pH 5.5, 140 mM NaCl, and 0.2 mg/ml polysorbate 80.It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Another aspect of the present invention provides kits comprising ananti-ErbB2 antibody or antibody portion of the invention or acomposition comprising such an antibody. A kit may include, in additionto the antibody or composition, diagnostic or therapeutic agents. A kitcan also include instructions for use in a diagnostic or therapeuticmethod. In another embodiment, the kit includes the antibody or acomposition comprising it and a diagnostic agent that can be used in amethod described below. In another preferred embodiment, the kitincludes the antibody or a composition comprising it and one or moretherapeutic agents that can be used in a method described below.

Diagnostic Methods of Use

In another aspect, the invention provides diagnostic methods. Theanti-ErbB2 antibodies can be used to detect ErbB2 in a biological samplein vitro or in vivo. In one embodiment, the invention provides a methodfor diagnosing the presence or location of an ErbB2-expressing cells ina subject in need thereof, comprising the steps of administering theantibody into the subject, determining the expression of ErbB2 in thesubject by localizing where the antibody has bound, comparing theexpression in the subject with that of a normal reference subject orstandard, and diagnosing the presence or location of the cells. Theanti-ErbB2 antibodies may also be used as a marker of proliferation.

The anti-ErbB2 antibodies can be used in a conventional immunoassay,including, without limitation, an ELISA, an RIA, flow cytometry, tissueimmunohistochemistry, Western blot or immunoprecipitation. Theanti-ErbB2 antibodies of the invention can be used to detect ErbB2 fromhumans. In another embodiment, the anti-ErbB2 antibodies can be used todetect ErbB2 from cynomolgus monkeys or rhesus monkeys. In anotherembodiment, the anti-ErbB2 antibodies can be used to detect ErbB2 fromrodents, such as mice and rats.

The invention provides a method for detecting ErbB2 in a biologicalsample comprising contacting the biological sample with an anti-ErbB2antibody of the invention and detecting the bound antibody. In oneembodiment, the anti-ErbB2 antibody is directly labeled with adetectable label. In another embodiment, the anti-ErbB2 antibody (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the anti-ErbB2 antibody is labeled. As is well known toone of skill in the art, a second antibody is chosen that is able tospecifically bind the particular species and class of the firstantibody. For example, if the anti-ErbB2 antibody is a human IgG, thenthe secondary antibody could be an anti-human-IgG. Other molecules thatcan bind to antibodies include, without limitation, Protein A andProtein G, both of which are available commercially, e.g., from PierceChemical Co.

Suitable labels for the antibody or secondary antibody have beendisclosed supra, and include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S and ³H.

In other embodiments, ErbB2 can be assayed in a biological sample by acompetition immunoassay utilizing ErbB2 standards labeled with adetectable substance and an unlabeled anti-ErbB2 antibody. In thisassay, the biological sample, the labeled ErbB2 standards and theanti-ErbB2 antibody are combined and the amount of labeled ErbB2standard bound to the unlabeled antibody is determined. The amount ofErbB2 in the biological sample is inversely proportional to the amountof labeled ErbB2 standard bound to the anti-ErbB2 antibody.

One can use the immunoassays disclosed above for a number of purposes.For example, the anti-ErbB2 antibodies can be used to detect ErbB2 incultured cells. In another embodiment, the anti-ErbB2 antibodies areused to determine the amount of ErbB2 on the surface of cells that havebeen treated with various compounds. This method can be used to identifycompounds that modulate ErbB2 protein levels. According to this method,one sample of cells is treated with a test compound for a period of timewhile another sample is left untreated. If the total level of ErbB2 isto be measured, the cells are lysed and the total ErbB2 level ismeasured using one of the immunoassays described above. The total levelof ErbB2 in the treated versus the untreated cells is compared todetermine the effect of the test compound.

A preferred immunoassay for measuring total ErbB2 levels is flowcytometry or immunohistochemistry. If the cell surface level of ErbB2 isto be measured, the cells are not lysed, and the cell surface levels ofErbB2 are measured using one of the immunoassays described above. Apreferred immunoassay for determining cell surface levels of ErbB2includes the steps of labeling the cell surface proteins with adetectable label, such as biotin or ¹²⁵I, immunoprecipitating the ErbB2with an anti-ErbB2 antibody and then detecting the labeled ErbB2.

Another preferred immunoassay for determining the localization of ErbB2,e.g., cell surface levels, is by using immunohistochemistry. A preferredimmunoassay to detect cell surface levels of ErbB2 includes binding ofan anti-ErbB2 antibody labeled with an appropriate fluorophore, such asfluorescein or phycoerythrin, and detecting the primary antibody usingflow cytometry. In another embodiment, the anti-ErbB2 antibody isunlabeled and a second antibody or other molecule that can bind theanti-ErbB2 antibody is labeled Methods such as ELISA, RIA, flowcytometry, Western blot, immunohistochemistry, cell surface labeling ofintegral membrane proteins and immunoprecipitation are well known in theart. See, e.g., Harlow and Lane, supra. In addition, the immunoassayscan be scaled up for high throughput screening in order to test a largenumber of compounds for either activation or inhibition of ErbB2.

The anti-ErbB2 antibodies of the invention also can be used to determinethe levels of ErbB2 in a tissue or in cells derived from the tissue. Inone embodiment, the anti-ErbB2 antibodies are used to determine theinfiltration of ErbB2-expressing cells into tissues that either do notexpress ErbB2 or that express it at reduced levels compared to theinfiltrating cells. In some embodiments, the tissue is a diseasedtissue. In some embodiments, the tissue is a tissue biopsy. In someembodiments of the method, a tissue or a biopsy thereof is excised froma subject. The tissue or biopsy is then used in an immunoassay todetermine, e.g., total ErbB2 levels, cell surface levels of ErbB2 orlocalization of ErbB2 by the methods discussed above. Such methods canbe used to determine whether a tissue expresses high levels of ErbB2,which could be indicative that the tissue is a target for treatment withanti-ErbB2 antibody.

The antibodies of the present invention also can be used in vivo toidentify tissues and organs that express ErbB2. In some embodiments, theanti-ErbB2 antibodies are used to identify ErbB2-expressing cells. Oneadvantage of using the human anti-ErbB2 antibodies of the presentinvention is that they may safely be used in vivo without eliciting asubstantial immune response to the antibody upon administration, unlikeantibodies of non-human origin or with humanized or chimeric antibodies.The method comprises the steps of administering a detectably labeledanti-ErbB2 antibody or a composition comprising them to a subject inneed of such a diagnostic test and subjecting the subject to imaginganalysis to determine the location of the ErbB2-expressing tissues.Imaging analysis is well known in the medical art, and includes, withoutlimitation, x-ray analysis, magnetic resonance imaging (MRI) or computedtomography (CT). The antibody can be labeled with any agent suitable forin vivo imaging, for example a contrast agent, such as barium, which canbe used for x-ray analysis, or a magnetic contrast agent, such as agadolinium chelate, which can be used for MRI or CT. Other labelingagents include, without limitation, radioisotopes, such as ⁹⁹Tc. Inanother embodiment, the anti-ErbB2 antibody will be unlabeled and willbe imaged by administering a second antibody or other molecule that isdetectable and that can bind the anti-ErbB2 antibody. In anotherembodiment, a biopsy is obtained from the subject to determine whetherthe tissue of interest expresses ErbB2.

In some embodiments, the detectably labeled anti-ErbB2 antibodycomprises a fluorophore. In certain embodiments, the fluorophore isselected from a near-infrared fluorescent dye, dinitrophenyl,fluorescein and derivatives thereof, rhodamine, derivatives ofrhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine, Texas red, Rhodamine green, Oregon green, Cascadeblue, phycoerythrin, CY3, CY5, CY2, CY7, coumarin, infrared 40, MR 200,IRD 40, Alexa Fluor, Cascade Blue, Tetramethylrhodamine, Pacific Blue,SYBR, and BODIPY. In another embodiment, the fluorophore includes one ofthe following compounds with their emission maxima indicated in nm inparenthesis, Cy2™ (506), GFP (Red Shifted) (507), YO-PRO®-1 (509),YOYO®-1 (509), Calcein (517), FITC (518), Fluor X® (519), Alexa® (520),Rhodamine 110 (520), 5-FAM (522), Oregon Green® 500 (522), Oregon Green®488 (524), RiboGreen® (525), Rhodamine Green® (527), Rhodamine 123(529), Magnesium Green® (531), Calcium Green® (533), TO-PRO®-1 (533),TOTO®-1 (533), JOE (548), BODIPY 12 530/550 (550), Dil (565), BODIPY®(568), BODIPY® 558/568 (568), BODIPY® 564/570 (570), Cy3® (570), Alexa®546 (570), TRITC (572), Magnesium Orange® (575), Phycoerythrin R&B(575), Rhodamine Phalloidin (575), Calcium Orange® (576), Pyronin Y(580), Rhodamine B (580), TAMRA (582), Rhodamine Red® (590), Cy3.5®(596), ROX (608), Calcium Crimsom™ (615), Alexa® 594 (615), Texas Red®(615), Nile Red (628), YO-PRO®-3 (631), YOYO®-3 (631), R-phycocyanin(642), C-Phycocyanin (648), TO-PRO®-3 (660), TOTO®-3 (660), DiD DilC (5)(665), Cy5™ (670), Thiadicarbocyanine (671) and Cy5.5 (694).

Therapeutic Methods of Use

In another embodiment, the invention provides a method for inhibitingErbB2 activity by administering a targed binding agent and/or ananti-ErbB2 antibody to a subject in need thereof. In another embodiment,the anti-ErbB2 antibody is a human, chimeric or humanized antibody. Inanother preferred embodiment, the ErbB2 is human and the subject is ahuman subject. Alternatively, the subject may be a mammal that expressesan ErbB2 with which the targed binding agent and/or anti-ErbB2 antibodycross-reacts. The targed binding agent and/or antibody may beadministered to a non-human mammal expressing ErbB2 with which theantibody cross-reacts (i.e. a cynomologus monkey) for veterinarypurposes or as an animal model of human disease. Such animal models maybe useful for evaluating the therapeutic efficacy of antibodies of thisinvention.

In one embodiment, the invention provides methods of treating orpreventing an ErbB2-mediated disorder in a subject by administering tothe subject a therapeutically-effective amount of a targed binding agentand/or an anti-ErbB2 antibody of the invention. As used herein, the term“an ErbB2-mediated disorder” is intended to include diseases and otherdisorders in which the presence of high or increased levels of ErbB2expression or activity in a subject suffering from the disorder havebeen shown to be, or are suspected of being, either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Such disorders may be evidenced, for example,by an increase in expression or the levels of ErbB2 on the cell surfacein the affected cells or tissues of a subject suffering from thedisorder, or by an increase in an ErbB2-mediated activity in a celltype, such as in a cancer cell, that contributes to the pathology of thedisorder or that contributes to the worsening of the disorder. Theincrease in ErbB2 levels may be detected, for example, using ananti-ErbB2 antibody. An increase in ErbB2 activity may be detected byincreased phosphorylation of ErbB2, activation of the MAPK pathway,activation of the p38-TSP-1 pathway, activation of the PI3K pathway,inhibition of CDC2, and combinations thereof.

Another aspect of the invention provides a method for inhibitingproliferation of a cancer cell, expressing ErbB2, in a subject in needthereof, the method comprising the step of administering to said subjecta targed binding agent and/or an anti-ErbB2 antibody or antigen-bindingportion thereof, wherein said targed binding agent or antibody orportion inhibits ErbB2. Another aspect provides a method for inhibitingan ErbB2 activity in a cell expressing ErbB2, comprising contacting thecell with a targed binding agent, an anti-ErbB2 antibody orantigen-binding portion thereof, wherein the ErbB2 activity in the cellis selected from the group consisting of (a) phosphorylation of ErbB2;(b) activation of the MAPK pathway; (c) activation of the p38-TSP-1pathway; (d) activation of the PI3K pathway; (e) inhibition of CDC2; and(f) combinations thereof. In another embodiment, the cell is in asubject.

The targed binding agent and/or the antibody may be administered once,but more preferably is administered multiple times. The targed bindingagent and/or the antibody may be administered from three times daily toonce every six months or longer. The administering may be on a schedulesuch as three times daily, twice daily, once daily, once every two days,once every three days, once weekly, once every two weeks, once everymonth, once every two months, once every three months and once every sixmonths. The targed binding agent and/or antibody may also beadministered continuously via a minipump. The targed binding agentand/or antibody may be administered via an oral, mucosal, buccal,intranasal, inhalable, intravenous, subcutaneous, intramuscular,intraperitoneal, intraocular, intraspinal, parenteral, intramucosal ortopical route. The targed binding agent and/or antibody may beadminstered locally or systemically.

The therapeutic compositions comprising a targed binding agent and/or aanti-ErbB2 antibodies may be administered to the subject, for example,orally, nasally, vaginally, buccally, rectally, via the eye, or via thepulmonary route, in a variety of pharmaceutically acceptable dosingforms, which will be familiar to those skilled in the art.

For example, the anti-ErbB2 antibodies may be administered via the nasalroute using a nasal insufflator device. Example of these are alreadyemployed for commercial powder systems intended for nasal application(e.g. Fisons Lomudal System). Details of other devices can be found inthe pharmaceutical literature (see for example Bell, A. IntranasalDelivery devices, in Drug Delivery Devices Fundamentals andApplications, Tyle P. (ed), Dekker, New York, 1988).

The anti-ErbB2 antibodies can be administered to the vagina in a freezedried powder formulation. Anti-ErbB2 antibodies may be administered in avaginal applicator and once in the vagina, the formulation comprisingthe anti-ErbB2 antibodies are released by pressing a syringe-type pistonor similar release mechanism on the applicator. Alternatively, theanti-ErbB2 antibodies may be formulated as a powder using a powderdevice, formulated into a vagina suppository or pessary or vaginaltablet or vaginal gel.

The anti-ErbB2 antibodies can also be administered to the eye in a gelformulation. For example, before administration, a formulationcontaining the anti-ErbB2 antibodies may be conveniently contained in atwo compartment unit dose container, one compartment containing afreeze-dried anti-ErbB2 antibody preparation and the other compartmentcontaining normal saline. Prior to application, the two compartments aremixed and a gel is formed, which is then administered to the eye.

Other delivery routes for the anti-ErbB2 antibodies include via thepulmonary route using a powder inhaler or metered dose inhaler, via thebuccal route formulated into a tablet or a buccal patch, via the rectalroute formulated into suppositories; and via the oral route in the formof a tablet, a capsule or a pellet (which compositions may administeragent via the stomach, the small intestine or the colon), all of whichmay be formulated in accordance with techniques which are well known tothose skilled in the art.

The antibody will generally be administered as part of a pharmaceuticalcomposition as described supra. The dosage of antibody will generally bein the range of 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, morepreferably 1-20 mg/kg, and even more preferably 1-10 mg/kg. The serumconcentration of the antibody may be measured by any method known in theart.

Co-administration of the antibody with an additional therapeutic agent(combination therapy) encompasses administering a pharmaceuticalcomposition comprising the anti-ErbB2 antibody and the additionaltherapeutic agent as well as administering two or more separatepharmaceutical compositions, one comprising the anti-ErbB2 antibody andthe other(s) comprising the additional therapeutic agent(s). Further,although co-administration or combination therapy generally means thatthe antibody and additional therapeutic agents are administered at thesame time as one another, it also encompasses instances in which theantibody and additional therapeutic agents are administered at differenttimes. For instance, the antibody may be administered once every threedays, while the additional therapeutic agent is administered once daily.Alternatively, the antibody may be administered prior to or subsequentto treatment of the disorder with the additional therapeutic agent, forexample after a subject has failed therapy with the additional agent.Similarly, administration of the anti-ErbB2 antibody may be administeredprior to or subsequent to other therapy, such as immunotherapy.

The antibody and one or more additional therapeutic agents (thecombination therapy) may be administered once, twice or at least theperiod of time until the condition is treated, palliated or cured.Preferably, the combination therapy is administered multiple times. Thecombination therapy may be administered from three times daily to onceevery six months. The administering may be on a schedule such as threetimes daily, twice daily, once daily, once every two days, once everythree days, once weekly, once every two weeks, once every month, onceevery two months, once every three months and once every six months, ormay be administered continuously via a minipump. The combination therapymay be administered via an oral, mucosal, buccal, intranasal, inhalable,intravenous, subcutaneous, intraocular, intraspinal, intraperitoneal,intramuscular, parenteral, intratumor or topical route.

In a still further embodiment, the anti-ErbB2 antibody is labeled with aradiolabel, an immunotoxin or a toxin, or is a fusion protein comprisinga toxic peptide. The anti-ErbB2 antibody or anti-ErbB2 antibody fusionprotein directs the radiolabel, immunotoxin, toxin or toxic peptide tothe ErbB2-expressing cell. In another embodiment, the radiolabel,immunotoxin, toxin or toxic peptide is internalized after the anti-ErbB2antibody binds to the ErbB2 on the surface of the cell.

Gene Therapy

The nucleic acid molecules of the present invention can be administeredto a subject in need thereof via gene therapy. The therapy may be eitherin vivo or ex vivo. In another embodiment, nucleic acid moleculesencoding both a heavy chain and a light chain are administered to asubject. In a more preferred embodiment, the nucleic acid molecules areadministered such that they are stably integrated into chromosomes of Bcells because these cells are specialized for producing antibodies. Inanother embodiment, precursor B cells are transfected or infected exvivo and re-transplanted into a subject in need thereof. In anotherembodiment, precursor B cells or other cells are infected in vivo usinga virus known to infect the cell type of interest. Typical vectors usedfor gene therapy include liposomes, plasmids and viral vectors.Exemplary viral vectors are retroviruses, adenoviruses andadeno-associated viruses. After infection either in vivo or ex vivo,levels of antibody expression can be monitored by taking a sample fromthe treated subject and using any immunoassay known in the art ordiscussed herein.

In another embodiment, the gene therapy method comprises the steps ofadministering an isolated nucleic acid molecule encoding the heavy chainor an antigen-binding portion thereof of an anti-ErbB2 antibody andexpressing the nucleic acid molecule. In another embodiment, the genetherapy method comprises the steps of administering an isolated nucleicacid molecule encoding the light chain or an antigen-binding portionthereof of an anti-ErbB2 antibody and expressing the nucleic acidmolecule. In a more preferred method, the gene therapy method comprisesthe steps of administering of an isolated nucleic acid molecule encodingthe heavy chain or an antigen-binding portion thereof and an isolatednucleic acid molecule encoding the light chain or the antigen-bindingportion thereof of an anti-ErbB2 antibody of the invention andexpressing the nucleic acid molecules. The gene therapy method may alsocomprise the step of administering another therapeutic agent, such as aanti-cancer agent.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

Example 1 Immunization and Titering

Immunization

Recombinant human ErbB2-ECD/Fcγ1 fusion protein containing theextracellular domain of human ErbB2 and the Fc region of human IgG1 wasobtained from R&D Systems, Inc. (Minneapolis, Minn. catalog #1129-ER/CF)for use as immunogen. Monoclonal antibodies against ErbB2 were developedby sequentially immunizing XenoMouse® mice (XenoMouse strain XM3B-3,Abgenix, Inc. Fremont, Calif.) via footpad route injections. The firstinjection was with 10 μg recombinant human ErbB2-ECD/Fcγ1 in TitermaxGold (Sigma, catalog #T2684, lot #K1599) per mouse. The following 10boosts were with 10 μg recombinant human ErbB2-ECD/Fcγ1 in 15 μl of qCpG(ImmunEasy Mouse Adjuvant, catalog #303101; Qiagen), admixed with 5 μlof Adju-Phos (aluminum phosphate gel, Catalog #1452-250, HCl Biosector)per mouse. The total volume of each injection was 50 μl per mouse, 25 μlper footpad. The mice were immunized twice weekly for 5 weeks and fusionwas performed on day 39.

Selection of Animals for Harvest by Titer

The immunized XenoMouse mice were bled after the 8th boost, andanti-ErbB2 antibody titers in the sera were determined by FACS(Fluorescence-Activated Cell Sorter) analysis.

For this purpose, a human ErbB2 expression vector was constructed andmouse pre-B B300.19 cells were transfected to express human ErbB2protein. Human ErbB2 cDNA was derived by RT-PCR from human epidermoidcarcinoma A431 cells (ATCC, catalog #CRL-1555) and cloned into thepCR3.1 expression vector (invitrogen, catalog #K3000) through HindIIIand Nod endonuclease restriction cleavage sites. The expression vectorcontained an insert of 3768 bp encoding the full length human ErbB2. Theabove plasmid was transfected into B300.19 cells using electroporationmethod. Stable B300.19 clones expressing hErbB2 protein were selected inthe presence of puromycin (2.5 ug/ml) and then screened by FACS with achimeric anti-hErbB2 antibody (referred to herein as 2C4, made asdetailed in Cancer Immunol. Immunotherapy (2006) 55:717-727 entitled“Humanization of a recombinant monoclonal antibody to produce atherapeutic HER dimerization inhibitor, pertuzumab”) followed by goatanti-mouse IgG PE (Caltag, catalog #M30004-4). B300.19/hErbB2 clone #44,giving the highest Geomean in FACS, was selected for sera titerdetermination.

Sera from immunized and bled mice were titrated in FACS buffer (PBS with2% FBS) at 1:50, 1:250 or 1:1250 dilutions. B300.19/hErbB2 clone #44cells (positive cells) and B300.19 parental cells (negative cells) wereincubated with serially diluted sera for 1 hour, and then withCy5-conjugated Goat anti-human IgG (Jackson ImmunoResearch Labs/JIR,catalog #109-176-098) for another 30 minutes. Anti-ErbB2 mAb 2C4 wasused as a positive control while an anti-KLH G1 antibody generated inhouse (Gmix) was used as a G1 isotype control. After extensive washing,cells were re-suspended in FACS buffer and analyzed on a BD FACSinstrument. Geomean of each sample was determined after data analysisand is shown in Table 2 below. The ratio of Geomean on ErbB2 positivecells over Geomean on ErbB2 negative cells correlates with the specificbinding ability to ErbB2. The negative controls, including G1 isotypecontrol anti-KLH Gmix, secondary control antibodies goat anti-mouse IgGCy5 (JIR, catalog #115-176-071) and goat anti-human IgG PE alone, gave aGeomean ratio of below 1. While the positive control mAb 2C4 and serafrom all 10 immunized mice gave ratios between 2.98 and 7.55, thereforeall mice developed humoral immune response to human ErbB2.

TABLE 2 Serum titers: 10 mice (XM3B-3 strain) GeoMeans Samples Assay poscells neg cells Geomean Mouse ID dilution GeoMean GeoMean Ratio 5152-11:50 324 62.2 5.21 1:250 251 45.1 5.57 1:1250 177 26.5 6.68 5152-2 1:50308 75 4.11 1:250 209 39.9 5.24 1:1250 146 23.4 6.24 5152-3 1:50 30485.3 3.56 1:250 199 40.1 4.96 1:1250 157 21.1 7.44 5152-4 1:50 331 1112.98 1:250 227 42.8 5.30 1:1250 166 22 7.55 5152-5 1:50 196 55.1 3.561:250 142 26.4 5.38 1:1250 91.7 16.2 5.66 5152-6 1:50 214 66.9 3.201:250 129 33.1 3.90 1:1250 92.4 17.5 5.28 5152-7 1:50 278 88.1 3.161:250 157 38.1 4.12 1:1250 122 20.5 5.95 5152-8 1:50 240 58.3 4.12 1:250168 27.2 6.18 1:1250 94.3 16 5.89 5152-9 1:50 208 46.2 4.50 1:250 13724.1 5.68 1:1250 89.9 15.9 5.65 5152-10 1:50 256 82.9 3.09 1:250 15740.5 3.88 1:1250 131 22.7 5.77

Example 2 Recovery of Lymphocytes, B-Cell Isolations, Fusions andGeneration of Hybridomas

Lymph nodes (LN) were harvested from the immunized mice and processedinto 3 ml sterile FASC buffer (PBS, 2% FBS). The LN cells were filteredthrough a 40 μm cell filter, spun down at 400 g for 3 minutes andresuspended in 3 ml fresh FACS buffer. The cells were counted, and thenbiotinylated antibodies against CD90 (Pharmingen, catalog #553002), CD4(Pharmingen, catalog #553728), CD8 (Pharmingen, catalog #553029) and IgM(Pharmingen, catalog #555781) were added. The cells and the aboveantibodies were mixed gently and incubated for 10 minutes on ice. Cellswere spun down again and washed once with FACS buffer. SA Dynal beads(M-280) were added to cells at a ratio of 4:1 beads to target cells, andincubated at room temperature for 12 minutes with rotating. Thecells/beads in 15 ml tubes were placed in the magnetic field of theDynal magnet for 2 minutes. The supernatant containing the IgM-fractionwas transferred to a fresh tube and the magnet step was repeated onemore time. The cell supernatant was transferred to a final tube andIgM-cells were counted and aliquoted for fusion.

The fusion was performed by mixing washed enriched B cells from aboveand non-secretory myeloma P3X63Ag8.653 cells purchased from ATCC(catalog #CRL 1580) (Kearney et al, J. Immunol. 123, 1979, 1548-1550) ata ratio of 1:1. The cell mixture was gently pelleted by centrifugationat 800×g. After complete removal of the supernatant, the cells weretreated with 2-4 ml of Pronase solution (CalBiochem, cat. #53702; 0.5mg/ml in PBS) for no more than 2 minutes. Then 3-5 ml of FBS was addedto stop the enzyme activity and the suspension was adjusted to 40 mltotal volume using electro cell fusion solution (ECFS: 0.3M Sucrose, 0.1mM Magnesium Acetate, 0.1 mM Calcium Acetate, all from Sigma). Thesupernatant was removed after centrifugation and the cells wereresuspended in 40 ml ECFS. This wash step was repeated and the cellsagain were resuspended in ECFS to a concentration of 2×10⁶ cells/ml.

Electro-cell fusion was performed using a fusion generator (modelECM2001, Genetronic, Inc., San Diego, Calif.), according to the standardinstrument settings. After ECF, the cell suspensions were carefullyremoved from the fusion chamber and transferred into a sterile tubecontaining the same volume of Hybridoma Culture Medium containing DMEM(JRH Biosciences), 15% FBS (Hyclone), supplemented with L-glutamine,Penicillin/Streptomycin, OPI (oxaloacetate, pyruvate, bovine insulin)(all from Sigma) and IL-6 (Boehringer Mannheim). The cells wereincubated for 15-30 minutes at 37° C., and then centrifuged at 400×g(1000 rpm) for five minutes. The cells were gently resuspended inHybridoma Selection Medium (Hybridoma Culture Medium supplemented with0.5×HA (Sigma, cat. #A9666)), based on a final plating of 2×10⁵ B cellstotal per 96-well plate and 200 μl per well. The cells were mixed gentlyand pipetted into 96-well plates and allowed to grow. On day 7 or 10,one-half the medium was removed, and the cells were re-fed withHybridoma Selection Medium.

Example 3 Screening of Antibodies by FMAT/FACS

After 14 days of culture, hybridoma supernatants were screened forErbB2-specific antibodies by FMAT (Fluorometric Microvolume AssayTechnology). Briefly, 4275 cells of B300.19/hErbB2 (positive cells) orB300.19 (ErbB2-negative cells) were mixed with 400 ng/ml Cy5 Goatanti-human IgG (JIR, catalog #109-176-098) in 15 μl of FACS buffer andthen incubated with 15 μl of hybridoma supernatants for 3 hours at roomtemperature. Positive control antibody was anti-hErbB2 (2C4), which wasdetected with Cy5 goat anti-mouse IgG gamma (JIR, catalog #115-176-071)or Goat anti-mouse IgG-Biot (Southern Biotechnology/SB catalog #1030-08,400 ng/ml) in combination with SA-Cy5 (JIR catalog #016-170-084, 350ng/ml). An anti-KLH G1 antibody (Gmix, in-house) was used as an isotypecontrol. The plates were read on FMAT 8100 HTS systems from AppliedBiosystems. Both fluorescence signals and counts were determined afterdata analysis and 362 positive hybridomas that showed binding toErbB2-positive cells but not to negative cells were identified.

The 362 positive supernatants in the FMAT screening were furtherscreened by FACS in two sets, one for hIgG heavy chain detection and theother for human Ig kappa light chain detection, to demonstrate fullyhuman composition for both Ig gamma and Ig kappa chains. 2.5×10⁵B300.19/hErbB2 cells or B300.19 parental cells were incubated withhybridoma supernatants diluted at 1:2 in FACS buffer for 1 hour at 4° C.and then washed with PBS. Cells were then incubated with goat anti-humangamma Cy5 for 1 hour at 4° C. for Ig gamma detection, or with goatanti-human kappa PE (SB catalog #2063-09) for 1 hour at 4° C. for Igkappa detection. After washing, cells were fixed in 1%paraformaldehyde/PBS before FACS analysis. Pooled sera at 1:50 dilutionwas used as a positive control while 1:10 diluted Gmix (anti-KLH IgG)was used as a negative isotype control in the assay. The ratio of theGeomean values between positive and negative cells was tabulated andhybridomas giving ratios over 1.95 were considered hits. A total of 152fully human anti-hErbB2 IgG/kappa antibodies were confirmed in thisscreen.

Example 4 Inhibition of Heregulin-β Induced ERBB2 Phosphorylation inMCF7 Cells

ErbB2 is tyrosine phosphorylated upon activation through dimerizationwith other ErbB family members, such as ErbB3. Heregulin-β binding toErbB3 can induce ErbB2 tyrosine phosphorylation in human breastadenocarcinoma MCF7 cells, which express both ErbB2 and ErbB3. Toidentify antibodies that block ErbB2 activation, we screened 152 humanErbB2-binding antibodies in cell-based ErbB2 phosphorylation assays.

MCF7 cells were seeded at 25,000 cells/well in 96 well plates andcultured in full growth media (10% FCS) overnight. The next day, cellculture plates were washed once with PBS and culture media was replacedwith phenol red-free and serum-free media. Cells were serum-starvedovernight, and then incubated with 25 μl of hybridoma supernatants mixedwith 25 μl phenol red-free medium for 1 hour before being treated with10 nM Heregulin-β for 10 minutes. Alternatively, cells were notserum-starved but incubated with hybridoma supernatants overnight (˜24hrs) before Heregulin-β treatment.

To prepare cell lysates, cells were washed in ice-cold PBS twice andincubated with 100 μl/well of lysis buffer (50 mM Tris-HCl p117.7, 1%Trixton X-100, 10% glycerol, 100 mM NaCl, 2.5 mM EDTA, 10 mM NaF, 40ug/ml PMSF, 1 uM Pepstatin, 0.5 ug/ml Leupeptin, 10 ug/ml SoybeanTrypsin inhibitor, 0.2 mM NaVO4, 1 mM NaMoO4, 5 mM b-glycerophosphate)at 4° C. for 30 minutes. Phosphor-ErbB2 level was measured using a humanphosphor-ErbB2 ELISA kit from R&D systems (human Phospho-ErbB2 DuoSetIC, Catalog #DYC1768), according to the protocol provided. Percentage ofinhibition was calculated based on the level of pErbB2 in un-stimulatedcells and Heregulin-stimulated cells in the absence of antibodies.

FIG. 1 illustrates the correlation of the neutralizing activities of the152 antibodies tested when they were pre-incubated with MCF7 cells for 1hr vs. 24 hr overnight. 51 out of 152 antibodies gave more than 30%inhibition when cells were pre-incubated with supernatants for 1 hour.These antibodies likely inhibit ErbB2 phosphorylation by blockingdimerization of ErbB2 with ErbB3. Significantly more antibodiesdemonstrated more than 30% inhibition when cells were pre-incubated withhybridoma supernatants overnight. Some of these may do so by othermechanism such as inducing ErbB2 down-regulation.

Example 5 Determination of Cross-Reactivity to CYNO ERBB2

To determine the species cross-reactivity of these antibodies tonon-human Primate cynomolgus ErbB2, CHO-K1 cells expressing cynomolgusErbB2 were generated. Briefly, the cyno ErbB2 cDNA was derived fromcynomolgus ovary tissues and cloned into pCR3.1 expression vectorthrough HindIII and XbaI restriction endonuclease sites. The expressionvector, cyno-ErbB2 (FL)/pCr3.1 containing an insert of 3767 bps, wastransfected into CHO-k1 cell using Lipofectamine 2000 (Invitrogen,catalog #11668) according to the protocol provided. Stable clones ofCHO-K1 expressing cyno-ErbB2 were selected in the presence of G418 at 1mg/ml. The expression was confirmed by FACS using mouse anti-humanc-ErbB2/c-neu (Ab-2) (Oncogene, catalog #OP14) and goat anti-mouseIgG-PE (Caltag, catalog #M30004-4).

CHO-K1/cyno E4rbB2 clone #4 was used to measure the cross-reactivity ofthe human ErbB2-binding antibodies. 200,000 cells of CHO-K1/cyno ErbB2clone #4 or parental CHO-K1 were incubated with 1:2 diluted hybridomasupernatants or 2 μg/ml positive control antibody Ab-2 (Oncogene,catalog #OP14) for 1 hour at 4° C. Cells were washed once with PBS, andthen incubated with secondary detection antibody 5 μg/ml of goatanti-human IgG Cy5 or goat anti-mouse IgG Cy5 for 1 hour at 4° C. Cellswere washed three times with PBS, fixed in 1% paraformaldehyde/PBS andthen analyzed by FACS. For each staining, the ratio of Geomean valuesbetween positive and negative cells was tabulated and a ratio above 1.95was considered positive. Eight antibodies were found not to cross-reactwith cyno ErbB2 and were excluded from the later analyses.

Example 6 High Antigen and Limited Antigen ELISAs

113 anti-ErbB2 antibodies that demonstrated cyno ErbB2 cross-reactivity,and also showed ≧30% inhibition of ErbB2 phosphorylation when incubatedwith cells for either 1 hr or overnight, were further characterized byhigh antigen (HA) and limited antigen (LA) ELISAs.

HA ELISA is performed with high concentrations of antigen coated onplate, and is a concentration-dependent reaction; while LA ELISA isconducted with limited amount of antigen coated on plate and thus is anaffinity dependent reaction. Relative affinity ranking of antibodies canbe achieved from HA/LA analysis.

Since the recombinant human ErbB2 ECD-Fcγ1 fusion protein cannot be usedfor this purpose, a human ErbB2 ECD-myc/His fusion protein was generatedin-house. The human ErbB2/ECD cDNA was derived from A431 cells andcloned into pSecTag2Hygro expression vector (Invitrogen, catalog#V910-20) through the NheI and XhoI restriction endonuclease cleavagesites. The expression vector, huErbB2 (ECD)/pSecTag2BHygro, contained aninsert size of 1956 bp for the ErbB2 (ECD) only and 2034 bp for thehErbB2 (ECD) plus c-myc/His tag. The plasmid was transiently transfectedinto 293T suspension cells using 293fectin (Invitrogen catalog#12347-019). The cells were treated with Sodium butyrate one day posttransfection to boost expression levels. 4 Days after transfection,supernatant was tested for ErbB2 (ECD) expression before purification.For ELISA detection, 1 μg/ml goat anti-ErbB2 (R&D systems, catalog#AF1129) was used to coat the plates, 1 μg/ml of mouse anti-ErbB2 (R&Dsystems, catalog #MAB 1129) was the primary detection antibody and thesecondary antibody was goat anti-mouse IgG-HRP (Caltag, catalog#M30107). Supernatant was harvested and concentrated 5 fold and thendialyzed into dialysis buffer (50 mM NaH2PO4, pH 8, 200 mM NaCl)overnight. Imidazole was added to the supernatant to a finalconcentration of 5 mM and the supernatant was incubated with 1/100^(th)volume of Ni-NTA superflow resin mixture (Qiagen, Cat#30430) for 3 hoursat RT. The resin was washed with a buffer containing 50 mM NaHPO4, 300mM NaCl and 20 mM Imidazole, and then with the elution buffer containing250 mM Imidazole, 50 mM NaHPO4 and 300 mM NaCl. The purified protein wasfinally dialyzed into PBS to preserve activity. The purified proteinhuErbB2 (ECD)/c-myc-His has 655 AAs, with a theoretical MW of 72.2 kDa,runs around 100 kDa on a 4-20% Tris-Glycine SDS-PAGE gel. The proteinidentity was confirmed by western blot using a mouse monoclonalanti-ErbB2 (R&D systems, Cat#MAB1129) followed by a secondary goatanti-mouse IgG-HRP (Caltag, cat #M30107).

For HA, 10 μg/ml human ErbB2 ECD-mycalis in PBS was coated to ELISAplates overnight at 4° C. For LA, 100, 500, 250, 125, 62, and 31 ng/mlof human ErbB2 ECD-myc/His was coated. Antigen-coated plates were washedthree times, blocked with 1% non-fat skim milk/PBS for at least 30minutes at room temperature, and then washed again three times. Eachhybridoma line sample was titrated in 1% non-fat skim-milk/PBS 1:3 for 7points starting from a 1:25 dilution. Serially diluted hybridoma samplesor controls (Herceptin®) were transferred to HA-coated plates and 1:25diluted hybridoma samples were transferred to LA-coated plates. Sampleswere incubated on plates at room temperature overnight for 18.5 hours.The next day, plates were washed three times and incubated with 50 μl of400 ng/ml immunopure Goat anti-human IgG (Fc)-HRP (Pierce catalog#31416) for 1 hour at room temperature. Then, the plates wereextensively washed and pat dried on paper towel to remove residual HRPin wells. HRP substrate TMB (enhanced K-blue TMB, Neogen catalog#308177) was added to each well and incubated for 30 min. The reactionwas stopped by addition of 1N HCl. Optical density at 450 nm was read ona microplate reader (Titerteck Multiskan Ascent).

The concentration of ErbB2-specific antibodies in hybridoma supernatantswas derived from a standard curve generated with known concentrations ofHerceptin® in HA ELISA. LA OD signal at 31 ng/ml antigen was plottedagainst antibody concentration from HA for each hybridoma sample, asshown in FIG. 2. The antibodies in the upper left corner have relativelyhigh affinities compared to the antibodies in the lower right corner inthis plot.

Example 7 Hybridoma Cloning

Based on the data from cyno cross-reactivity testing, inhibition ofErbB2 phosphorylation assays and LA/HA ELISAs, 31 hybridoma lines wereselected for cloning. Their activities are summarized in Table 3.

TABLE 3 Preliminary characterization data of hyrbidoma lines selectedfor cloning 1 hr pTyr 24 hr pTyr LINE inhibi- inhibi- Cyno Cross- HA AvgLA at ID tion % tion % reactivity (ug/ml) 31 ng/ml 1*14 103 100 YES 7.273.99 1*15 86 100 YES 0.61 2.31 1*18 85 96 YES 1.22 3.03 1*20 91 96 YES1.5 2.84 1*22 84 95 YES 1.06 1.61 1*37 96 96 YES 1.3 3.33 1*39 82 99 YES0.68 2.57 1*62 82 98 YES 0.51 2.8 1*96 97 99 YES 6.48 4.6 1*99 97 99 YES6.18 4.05 1*100 95 95 YES 2.85 3.54 1*108 90 100 YES 4.41 2.64 1*124 9497 YES 2.86 3.42 1*128 96 98 YES 5.86 3.83 1*140 97 97 YES 4.39 3.541*148 96 97 YES 3.29 3.35 1*149 83 89 YES 0.43 1.54 1*19 73 97 YES 0.41.77 1*24 77 99 YES 1.77 2.35 1*33 73 101 YES 0.9 2.07 1*41 26 86 YES1.3 3.2 1*43 43 82 YES 1.3 3.57 1*44 78 98 YES 3.64 2.38 1*69 57 72 YES0.34 1.6 1*71 27 65 YES 3.1 2.62 1*74 66 90 YES 0.20 1.22 1*79 71 93 YES0.26 1.77 1*95 58 95 YES 0.23 1.14 1*104 21 73 YES 1.7 2.36 1*107 57 82YES 0.25 1.15 1*111 78 98 YES 0.77 1.97

Cells in each hybridoma line were sorted on FACS Aria (BD) into 96 wellplates at 1 cell/well and cultured for about 2 weeks. Supernatants fromsingle clones were screened for ErbB2-binding activity by FMAT on BT474cells which express high level of ErbB2, and also for human Ig gamma andkappa chain composition, as well as presence of human IgM and mouse IgMby ELISA.

For FMAT, 6000 BT474 cells (ATCC) in 40 μl FACS buffer in 384 well FMATplates were incubated with 15 μl of supernatants for 2 hour at roomtemperature and then with 10 μl of 4.5 μg/ml goat-anti-human IgG Cy5 for6 hours at room temperature before reading on FMAT machine 8200. Bothfluorescence and counts data were analyzed. Herceptin® was used as apositive control in the screen.

For human antibody chain composition analysis, medium binding 96 wellplates (Coastar 3368) were coated with 2 μg/ml Goat anti-human IgG Fc inPBS overnight at 4° C., and blocked with 1% milk/PBS for 30 minutes atroom temperature, Supernatants were diluted 1:5 in 1% milk/PBS and addedto two coated ELISA plates, and incubated at room temperature for 1hour. Biotinylated goat anti-human Kappa (Vector catalog #BA3060) at 250ng/ml in 1% milk/PBS or biotinylated goat anti-human Lambda (SouthernBiotech catalog #2070-08) at 250 ng/ml in 1% milk/PBS were then addedand incubated for 1 hour at room temperature followed by incubation withStreptavidin Peroxidase conjugate at 1 μg/ml in 1% milk/PBS for 1 hourat room temperature. Plates were extensively washed between incubationsteps. 50 μl of Peroxidase substrate TMB was added and incubated for 30minutes at room temperature, and the enzyme reaction was quenched with50 μl of 1M HCL. Optical density was read at 450 nm on a microplatereader (Titerteck Multiskan Ascent).

We confirmed the absense of human IgM as follows. Medium binding 96 wellplates (Coastar 3368) were coated with 1 μg/ml Goat anti-human IgM inPBS overnight at 4° C., and blocked with 1% milk/PBS for 30 minutes atroom temperature. Supernatants were diluted 1:5 in 1% milk/PBS and addedto the coated ELISA plates, and incubated at room temperature for 1hour. Donkey anti-human 1gM POD (Accurate Chemical catalog #JNH035043)at 666 ng/ml in 1% milk/PBS was then added and incubated for 1 hour atroom temperature. Plates were developed by addition of POD substrate asdescribed above.

We confirmed the absense of human IgM as follows. Medium binding 96 wellplates were coated with 1 μg/ml Goat anti-mouse lambda (Southern Biotech1060-01) in PBS overnight at 4° C., and blocked with 1% milk/PBS for 30minutes at room temperature. Supernatants were diluted 1:5 in 1%milk/PBS and added to the coated ELISA plates, and incubated at roomtemperature for 1 hour. Goat anti-human IgG (Fe) POD (Pierce catalog#31413) at 400 ng/ml in 1% milk/PBS was then added and incubated for 1hour at room temperature. Plates were developed by addition of PODsubstrate as described before.

In the ELISAs described above, signal from wells with no primaryantibodies was used as background and samples giving signals that were 3times above the background were considered positive.

Monoclonal antibodies that demonstrated native binding to ErbB2 by FMATand that were positive for human gamma chain and kappa chain by ELISAswere derived from 20 hybridoma lines after cloning. Three subclones fromeach parental line were sequenced. Unless otherwise indicated all threesubclones were identical. For example, subclones 1.14.1, 1.14.2, and1.14.3 of parent clone 1.14 all had the same sequence and are referredto interchangably by the two-number parent name or the three-numbersubclone name. Eleven unique monoclonal antibodies were identified andfurther characterized in cell-based functional assays.

Example 8 Structural Analysis of ErbB2 Monoclonal Antibodies

The heavy chain and the light chain variable domains of the antibodieswere sequenced. The complete sequence information for the anti-ErbB2antibodies is provided in the Sequence Listing with nucleotide and aminoacid sequences for each gamma and kappa chain combination. The variable(V) regions of immunoglobulin chains are encoded by multiple germ lineDNA segments, which are joined into functional variable regions(V_(H)DJ_(H) or V_(I)A) during B-cell ontogeny. The variable heavysequences were analyzed to determine the VH family, the D-regionsequence and the J-region sequence. The sequences were then translatedto determine the primary amino acid sequence and compared to thegermline VH, D and J-region sequences to assess somatic hypermutations.The heavy variable light chain sequences were similarly analyzed.

Table 4 and Table 4(a) are tables comparing the antibody heavy chainregions to their cognate germ line heavy chain region. Table 5 is atable comparing the antibody kappa light chain regions to their cognategerm line light chain region. The difference between Tables 4 and 4(a)is the definition used to define the heavy chain CDR1s. The heavy chainCDR1s disclosed in Table 4(a) are of the Kabat definition.Alternatively, the CDR can be defined using an alternative definition soas to include the last four residues of the FR1 sequence as shown inTable 4.

Analysis of 20 individual antibodies specific to ErbB2 indicated thatsome of them are identical and only 11 unique monoclonal antibodies wereresulted from cloning 8 of the 11 antibodies are very similar insequence and were derived from the same germline VH and VK genes.

It should also be appreciated that where a particular antibody differsfrom its respective germline sequence at the amino acid level, theantibody sequence can be mutated back to the germline sequence. Suchcorrective mutations can occur at one, two, three or more positions, ora combination of any of the mutated positions, using standard molecularbiological techniques. By way of a non-limiting example, Table 4 showsthe heavy chain sequence of 1.20.1 differs from the correspondinggermline sequence at amino acid 33 by a T to an S in the CDR1 region.Thus, the amino acid or nucleotide sequence encoding the heavy chain of1.20.1 can be modified to change the T to an S to yield the germlinesequence at the site of the mutation. In another example, the heavychain sequence of 1.140.1 differs from the corresponding germlinesequence at amino acid 42 by an R to a G in the FR2 region. Thus, theamino acid or nucleotide sequence encoding the heavy chain of 1.140.1can be modified to change the R to a G to yield the germline sequence atthe site of the mutation.

By way of another non-limiting example, Table 5 shows that the lightchain sequence of 1.140.1 differs from the corresponding germlinesequence by a T to N mutation (mutation 1) in the FR1 region, by a F toL, F to Y and C to Y (mutations 2, 3, and 4) in the CDR1 region, by a Rto K and N to K in the FR2 region (mutations 5 and 6) and by a F to Y, Gto S, and S to T in the CDR3 region. Thus, the amino acid or nucleotidesequence encoding the light chain of 1.140.1 can be modified to changemutation 1 to yield the germline sequence at the site of mutation 1.Further, the amino acid or nucleotide sequence encoding the light chainof 1.140.1 can be modified to change mutation 2 to yield the germlinesequence at the site of mutation 2. Still further, the amino acid ornucleotide sequence encoding the light chain of 1.140.1 can be modifiedto change mutation 3 to yield the germline sequence at the site ofmutation 3. Still further again, the amino acid or nucleotide sequenceencoding the light chain of 1.140.1 can be modified to change mutation1, mutation 2, mutation 3, mutation 4, mutation 5 and mutation 6 toyield the germline sequence at these. Table 6 below illustrates theposition of such variations from the germline for 1.140. Each rowrepresents a unique combination of germline and non-germline residues atthe position indicated by bold type. Table 6 also applies to antibody1.39 as the light chain analysis for antibody 1.39 is identical to 1.140with respect to germline mutations. Moreover, the light chain analysisfor antibody 1.96 is identical to 1.140 with respect to germlinemutations except that the antibody 1.96 has a difference between theantibody sequence and the germline sequence in FR4. In this example, theL of the antibody sequence can be mutated back to the germline sequenceof an F, and this mutation can be combined with any of the combinationsshown in Table 6.

In one embodiment, the invention features modifying one or more of theamino acids in the CDR regions, i.e., CDR1, CDR2 and/or CDR3. In oneexample, the CDR3 of the heavy, light or both chains of an antibodydescribed herein is modified. Typically, the amino acid is substitutedwith an amino acid having a similar side chain (a conservative aminoacid substitution), back to germline, or can be substituted with anyappropriate amino acid such as an alanine or a leucine. In oneembodiment, the 1.24.3 CDR3, QQYSSPFT (SEQ ID NO: 48) can be modified atone or more amino acids back to for example by mutating the CDR3sequence to QQYYSPFT (SEQ ID NO:49).

In another embodiment, the invention features modifying the antibody toremove unpaired cysteines. Examples of unpaired cysteines appear inantibody 1.39.1 at amino acid 38, in antibody 1.96.1 at amino acid 38,and for antibody 1.140.1 at amino acid 38. These cysteines can bemutated back to germline, for example, mutating the C to Y or bymutating the C to any appropriate amino acid such as a serine.

TABLE 4 Heavy Chain Analysis SEQ Chain Name ID NO: V D J FR1 CDR1 FR2 54Germline EVQLVESGGGLVK GFTFSSYSMN WVRQAPGKGLEWVS PGGSLRLSCAAS 1.14.1 14VH3-21 D5-24 JH4B EVQLVESGGGLVK GFTFSSYSMN WVRQAPGKGLEWVS PGGSLRLSCAAS1.18.1 26 ″ ″ ″ EVQLVESGGGLVK GFTFSSYSMN WVRQAPGKGLEWVS PGGSLRLSCAAS1.20.1 = 30 ″ ″ ″ EVQLVESGGGLVK GFTFSSYTMN WVRQAPGKGLEWVS 1.19PGGSLRLSCAAS 1.24.3 = 38 ″ ″ ″ EVQLVESGGGLVK GFTFSSYSMN WVRQAPGKGLEWVS1.22.2 = PGGSLRLSCAAS 1.71.1 1.39.1 34 ″ ″ ″ EVQLVESGGGLVK GFTFSSYSMNWVRQAPGKGLEWVS PGGSLRLSCAAS 1.96.2 = 22 ″ ″ ″ EVQLVESGGGLVK GFTFSSYSMNWVRQAPGKGLEWVS 1.99 = PGGSLRLSCAAS 1.104 = 1.107 = 1.128 1.100.1 18 ″ ″″ EVQLVESGGGLVK GFTFSSYSMN WVRQAPGKGLEWVS PGGSLRLSCAAS 1.140.1 = 6 ″ ″ ″EVQLVESGGGLVK GFTFSSYSMN WVRQAPRKGLEWVS 1.124 = PGGSLRLSCAAS 1.148 57Germline EVQLVESGGGLVQ GFTFSSYWMS WVRQAPGKGLEWVA PGGSLRLSCAAS 1.43.1 =10 VH3-7 D5-24 JH6B EVQLVESGGGLVQ GFTFSSYWMH WVRQTPGKLGEWVA 1.41 =PGGSLRLSCAAS 1.22.1 59 Germline QVQLQESGPGLVK GGSISSGGYY WIRQHPGKLEWIGPSQTLSLTCTVS WS 1.44.1 2 VH4-31 D3-10 JH6B QVQLQESGPGLVK GGSISSGGYYWIRQHPGKGLEWIG PSQTLSLTCTVS WS 56 germline EVQLVESGGGLVQ GFTFSSYDMHWVRQATGKGLEWVS PGGSLRLSCAAS 1.71.2 = 42 VH3-13 D6-19 JH6B EVQLVESGGGLVQGFPFSSYDMH WVRQATGKGLEWVS 1.71.3 PGGSLRLSCTAS SEQ Chain Name ID NO: CDR2FR3 CDR3 FR4 54 SISSSSSYIY RFTISRDNAKNSL ##DGYNY WGQGTLVTVSS YADSVKGYLQMNSLRAEDTA #YFDY VYYCAR 1.14.1 14 SISSSSSYIY RFTISRDNAKNSL GGDGYNYWGQGTLVTVSS YADSVKG YLQMNSLRAEDTA YYFDY VYYCAR 1.18.1 26 SISSSSSYIYRFTISRDNAKNSL GGDGYNY WGQGTLVTVSS YADSVKG YLQMNSLSAEDTA YYFDY VYSCAR1.20.1 = 30 SISSSSSYIY RFTISRDNAKNSL GGDGYNY WGQGTLVTVSS 1.19 YADSVKGYLAMNSLSAEDTA YYFDY VYSCAR 1.24.3 = 38 SISSSSSYIY RFTISRDNAKNSL GGDGYNYWGQGTLVTVSS 1.22.2 = YADSVKG YLQMNSLRAEDTA YYFDY 1.71.1 VYYCAR 1.39.1 34SISSSSSYIY RFTISRDNAKNSL GGDGYNY WGQGTLVTVSS YADSVKG YLQMNSLRAEDTA YYFDYVYYCAR 1.96.2 = 22 SISSSSSYIY RFTISRDNAKNSL GGDGYNY WGQGTLVTVSS 1.99 =YADSVKG YLQMNSLRAEDTA YYDFY 1.104 = VYYCAR 1.107 = 1.128 1.100.1 18SISSSSSYIY RFTISRDNAKNSL GGDGYNY WGQGTLVTVSS YADSVKG YLQMNSLRAEDTA YYFDYVYYCAR 1.140.1 = 6 SISSSSSYIY RFTISRDNAKNSL GGDGYNY WGQGTLVTVSS 1.124 =YADSVKG YLQMNSLRAEDTA YYFDY 1.148 VYYCAR 57 NIKQDGSEKY RFTISRDNAKNSL###YGMDV WGQGTTVTVSS YVDSVKG YLQMNSLRAEDTA VYYCA# 1.43.1 = 10 NIKQDGSEKYRFTISRDNAKNSL FRDYGMDV WGQGTTVTVSS 1.41 = YVDSVKG HLQMNSLRAEDTA 1.22.1AYYCAS 59 YIYYSGSTYY RVTISVDTSKNQF ###ITMVR WGQGTTVTVSS NPSLKSSLKLSSVTAADTA GVYYYYYG VYYCAR MDV 1.44.1 2 YIYYSGSTYY RVTISVDTSKNQFEGPITIVR WVQGTTVTVSS NPSLKS SLKLSSVTAADTA GVYYYFYG VYYCAR MDV 56AIGTAGDTYY RFTISRENAKNSL #GYSS##Y WGQGTTVTVSS PGSVKG YLQMNSLRAGDTAYYYGMDV VYYCAR 1.71.2 = 42 AIGTAGDTFY RFTISRENAKNSL EGYSSGRY WGQGTTVTVSS1.71.3 PGSVKG YLAMNSLRAGDTA FYYGMDV VYYCAR

TABLE 4(a) Heavy Chain Analysis SEQ Chain Name ID NO: V D J FR1 CDR1 FR254 Germline EVQLVESGGGLVKPG SYSMN WVRQAPG GSLRLSCAASGFTFS KGLEWVS 1.14.114 VH3-21 D5-24 JH4B EVQLVESGGGLVKPG SYSMN EVRQAPG GSLRLSCAASGFTFSKGLEWVS 1.18.1 26 ″ ″ ″ EVQLVESGGGLVKPG SYSMN WVRQAPG GSLRLSCAASGFTFSKGLEWVS 1.20.1 = 30 ″ ″ ″ EVQLVESGGGLVKPG SYTMN WVRQAPG 1.19GSLRLSCAASGFTFS KGLEWVS 1.24.3 = 38 ″ ″ ″ EVQLVESGGGLVKPG SYSMN WVRQAPG1.22.2 = GSLRLSCAASGFTFS KGLEWVS 1.71.1 1.39.1 34 ″ ″ ″ EVQLVESGGGLVKPGSYSMN WVRQAPG GSLRLSCAASGFTFS KGLEWVS 1.96.2 = 22 ″ ″ ″ EVQLVESGGGLVKPGSYSMN WVRQAPG 1.99 = GSLRLSCAASGFTFS KGLEWVS 1.104 = 1.107 = 1.1281.100.1 18 ″ ″ ″ EVQLVESGGGLVKPG SYSMN WVRQAPG GSLRLSCAASGFTFS KGLEWVS1.140.1 = 6 ″ ″ ″ EVQLVESGGGLVKPG SYSMN WVRQAPR 1.124 = GSLRLSCAASGFTFSKGLEWVS 1.148 58 Germline EVQLVESGGGLVQPG SYWMS WVRQAPG GSLRLSCAASGFTFSKGLEWVA 1.43.1 = VH3-7 D5-24 JH6B EVQLVESGGGLVQPG SYWMH WVRQTPG 1.41 =GSLRLSCAASGFTFS KGLEWVA 1.22.1 10 Germline QVQLQESGPGLVKPS SGGYYWSWIRQHPG QTLSLTCTVSGGSIS KGLEWIG 1.44.1 2 VH4-31 D3-10 JH6BQVQLQESGPGLVKPS SGGYYWS WIRQHPG QTLSLTCTVSGGSIS KGLEWIG 56 GermlineEVQLVESGGGLVQPG SYDMH WVRQATG GSLRLSCAASGFTFS KGLEWVS 1.71.2 = 42 VH3-13D6-19 JH6B EVQLVESGGGLVQPG SYDMH WVRQATG 1.71.3 GSLRLSCTASGFPFS KGLEWVSSEQ Chain Name ID NO: CDR2 FR3 CDR3 FR4 54 SISSSSSYIY RFTISRDNAKNSLYL##DGYNY WGQGTLV YADSVKG QMNSLRAEDTAVYYC #YFDY TVSS AR 1.14.1 14SISSSSSYIY RFTISRDNAKNSLYL GGDGYNY WGQGTLV YADSVKG QMNSLRAEDTAVYYC YYFDYTVSS AR 1.18.1 26 SISSSSSYIY RFTISRDNAKNSLYL GGDGYNY WGQGTLV YADSVKGQMNSLSAEDTAVYSC YYFDY TVSS AR 1.20.1 = 30 SISSSSSYIY RFTISRDNAKNSLYLGGDGYNY WGQGTLV 1.19 YADSVKG QMNSLSAEDTAVYSC YYFDY TVSS AR 1.24.3 = 38SISSSSSYIY RFTISRDNAKNSLYL GGDGYNY WGQGTLV 1.22.2 = YADSVKGQMNSLRAEDTAVYYC YYFDY TVSS 1.71.1 AR 1.39.1 34 SISSSSSYIYRFTISRDNAKNSLYL GGDGYNY WGQGTLV YADSVKG QMNSLRAEDTAVYYC YYFDY TVSS AR1.96.2 = 22 SISSSSSYIY RFTISRDNAKNSLYL GGDGYNY WGQGTLV 1.99 = YADSVKGQMNSLRAEDTAVYYC YYFDY TVSS 1.104 = AR 1.107 = 1.128 1.100.1 18SISSSSSYIY RFTISRDNAKNSLYL GGDGYNY WGQGTLV YADSVKG QMNSLRAEDTAVYYC YYFDYTVSS AR 1.140.1 = 6 SISSSSSYIY RFTISRDNAKNSLYL GGDGYNY WGQGTLV 1.124 =YADSVKG QMNSLRAEDTAVYYC YYFDY TVSS 1.148 AR 58 NIKQDGSEKYRFTISRDNAKNSLYL #RDYGMD WGQGTTV YVDSVKG QMNSLRAEDTAVYYC V TVSS A#1.43.1 = NIKQDGSEKY RFTISRDNAKNSLHL FRDYGMD WGQGTTV 1.41 = YVDSVKGQMNSLRAEDTAAYYC V TVSS 1.22.1 AS 10 YIYYSGSTYY RFTISVDTSKNQFSL ###ITMVWGQGTTV NPSLKS KLSSVTAADTAVYYC RGVYYYY TVSS AR YGMDV 1.44.1 2 YIYYSGSTYYRVTISVDTSKNQFSL EGPITIV WGQGTTV NPSLKS KLSSVTAADTAVYYC RGVYYYF TVSS ARYGMDV 56 AIGTAGDTYY RFTSIRENAKNSLYL #GYSS## WGQGTTV PGSVKGQMNSLRAGDTAVYYC YYYYGMD TVSS AR V 1.71.2 = 42 AIGTAGDTFY RFTISRENAKNSLYLEGYSSGR WGQGTTV 1.71.3 PGSVKG QMNSLRAGDTAVYYC YFYYGMD TVSS AR V

TABLE 5 Light Chain Analysis SEQ Chain Name ID NO: V J FR1 CDR1 FR2 51Germline DIVMTQSPDSLAV KSSQSVLYSSNN WYQQKPGQ SLGERATINC KNYLA PPKLLIY1.14.1 16 B3 JK3 DIVMTQSPDSLAV KSSQSVFFRSNN WYQQRPGQ (VK4) SLGERATITCKNCLT PPNLLIY 1.18.1 28 ″ ″ DIVMTQSPGSLVV KSSQSVFFRSNN WYQQRPGQSLGERATITC KNCLA SPNLLIY 1.20.1 = 32 ″ ″ DIVMTQSPGSLVV KSSQSVFFRSNNWYQQRPGQ 1.19 SLGERATITC KNCLA SPNLLIY 1.24.3 = 40 ″ ″ DIVMTQFPDLSAVKSSQSVFFRSNN WYQQKPGQ 1.22.2 = SLDERATINC KNCLA PPNLLIY 1.71.1 1.39.1 36″ ″ DIVMTQSPDSLAV KSSQSVFFRSNN WYQQRPGQ SLGERATITC KNCLA PPNLLIY1.96.2 = 24 ″ ″ DIVNTQSPDSLAV KSSQSVFFRSNN WYQQRPGQ 1.99 = SLGERATITCKNCLA PPNLLFY 1.104 = 1.107 = 1.128 1.100.1 20 ″ ″ DIVMTQSPDSLAVKSSQSVFFRSNN WYQQRPGQ SLGERATITC KNCLA PPNLLIY 1.140.1 = 8 ″ ″DIVMTQSPDSLAV KSSQSVFFRSNN WYQQRPGQ 1.124 = SLGERATITC KNCLA PPNLLIY1.148 50 Germline DIQMTQSPSSLSA RASQGISNYLA WFQQKPGK SVGDRVTITC APKSLIY1.43.1 = 12 L1 JK5 DIQMTQSPSSLSA RASQGISNHLA WFQQKPGK 1.41 = (VK1)SVGDRVTITC APKSLIY 1.22.1 52 Germline DIVMTQTPLSLSV KSSQSLLHSDGKWYLQKPGQ TPGQPASISC TYLY PPQLLIY 1.44.1 4 A2 JK1 DIVMTQTPLSLSVKSSQSLLHSDGK WYLQKPGQ (VK2) TPGQPASISC TYLY PPQPLIY 53 GermlineDVVMTQSPLSLPV RSSQSLVYSDGN WFQQRPGQ TLGQPASISC TYLN SPRRLIY 1.71.1 = 44A1 JK4 DVVMTQSPLSLPV RSSQSLVYSDGN WFQQRPGQ 1.71.3 TLGQPASISC TYLNSPRRLIY SEQ Chain Name ID NO: CDR2 FR3 CDR3 FR4 51 WASTRES GVPDRFSGSGSGTQQYYSTPFT FGPGTKVDIK DFTLTISSLQAED VAVYYC 1.14.1 16 WASTRESGVPDRFSGSGSGT QQYFGSPFT FGPGTKVDIK DFTLTINNLQAED VAVYYC 1.18.1 28WASTRES GVPDRFSGSGSGT QQYFGSPFT FGPGTKVDIK DFTLTISSLQAED VAVYYC 1.20.1 =32 WASTRES GVPDRFSGSGSGT QQYFGSPFT FGPGTKVDIK 1.19 DFTLTISSLQAED VAVYYC1.24.3 = 40 WASTRES GVPDRFSGSGSGT QQYYSSPFT FGPGTKVDIK 1.22.2 =DFTLTISSLQAED 1.71.1 VAFYYC 1.39.1 36 WASSRES GVPDRFSGSGSGT QQYFGSPFTFGPGTKVDIK DFALTISSLQTED VAVYYC 1.96.2 = 24 WASTRES GVPDRFSGSGSGTQQYFGSPFT LGPGTKVDIK 1.99 = DFTLTISSLQAED 1.104 = VAVYYC 1.107 = 1.1281.100.1 20 WASTRES GVPDRFSGSGCGT QQYFGSPFT FGPGTKVDIK DFTLTISSLQAEDVAVYYC 1.140.1 = 8 WASTRES GVPDRFSGSGSGT QQYFGSPFT FGPGTKVDIK 1.124 =DFTLTISSLQAED 1.148 VAVYYC 50 AASSLQS GVPSRFSGSGSGT QQYNSYPIT FGQGTRLEIKDFTLTISSLQPED FATYYC 1.43.1 = 12 GASSLQT GVPSKFSGSGSGT QQYKGYPITFGQGTRLEIK 1.41 = DFTLTISSLQPED 1.22.1 FASYFC 52 EVSNRFS GVPDRFSGSGSGTMQSIQLPRT FGQGTKVEIK DFTLKISRVEAED VGVYYC 1.44.1 4 EVSNRFS GVPDRFSGSGSGTMQSKQLPRT FGQGTKVEIK DFTLKISRVEAED VGIYYC 53 KVSNWDS GVPDRFSGSGSGTMQGTHW##T FGGGTKVEIK DFTLKISRVEAED VGVYYC 1.71.1 = 44 KVSNWDSGVPDRFSGSGSGT MQGTHWPLT FGGGTKVEIK 1.71.3 DFTLKISRVEAED VGVYYC

TABLE 6 Exemplary Mutations of 1.140 (SEQ ID NO: 8) Light Chain toGermline at the indicated Residue Number 22 30 31 32 38 45 51 98 99 100N V L Y C K K Y S S N V L Y C K K Y S I N V L Y C K K Y G S N V L Y C KK Y G I N V L Y C K K F S S N V L Y C K K F S I N V L Y C K K F G S N VL Y C K K F G I N V L Y C K N Y S S N V L Y C K N Y S I N V L Y C K N YG S N V L Y C K N Y G I N V L Y C K N F S S N V L Y C K N F S I N V L YC K N F G S N V L Y C K N F G I N V L Y C R K Y S S N V L Y C R K Y S IN V L Y C R K Y G S N V L Y C R K Y G I N V L Y C R K F S S N V L Y C RK F S I N V L Y C R K F G S N V L Y C R K F G I N V L Y C R N Y S S N VL Y C R N Y S I N V L Y C R N Y G S N V L Y C R N Y G I N V L Y C R N FS S N V L Y C R N F S I N V L Y C R N F G S N V L Y C R N F G I N V L YY K K Y S S N V L Y Y K K Y S I N V L Y Y K K Y G S N V L Y Y K K Y G IN V L Y Y K K F S S N V L Y Y K K F S I N V L Y Y K K F G S N V L Y Y KK F G I N V L Y Y K N Y S S N V L Y Y K N Y S I N V L Y Y K N Y G S N VL Y Y K N Y G I N V L Y Y K N F S S N V L Y Y K N F S I N V L Y Y K N FG S N V L Y Y K N F G I N V L Y Y R K Y S S N V L Y Y R K Y S I N V L YY R K Y G S N V L Y Y R K Y G I N V L Y Y R K F S S N V L Y Y R K F S IN V L Y Y R K F G S N V L Y Y R K F G I N V L Y Y R N Y S S N V L Y Y RN Y S I N V L Y Y R N Y G S N V L Y Y R N Y G I N V L Y Y R N F S S N VL Y Y R N F S I N V L Y Y R N F G S N V L Y Y R N F G I N V L R C K K YS S N V L R C K K Y S I N V L R C K K Y G S N V L R C K K Y G I N V L RC K K F S S N V L R C K K F S I N V L R C K K F G S N V L R C K K F G IN V L R C K N Y S S N V L R C K N Y S I N V L R C K N Y G S N V L R C KN Y G I N V L R C K N F S S N V L R C K N F S I N V L R C K N F G S N VL R C K N F G I N V L R C R K Y S S N V L R C R K Y S I N V L R C R K YG S N V L R C R K Y G I N V L R C R K F S S N V L R C R K F S I N V L RC R K F G S N V L R C R K F G I N V L R C R N Y S S N V L R C R N Y S IN V L R C R N Y G S N V L R C R N Y G I N V L R C R N F S S N V L R C RN F S I N V L R C R N F G S N V L R C R N F G I N V L R Y K K Y S S N VL R Y K K Y S I N V L R Y K K Y G S N V L R Y K K Y G I N V L R Y K K FS S N V L R Y K K F S I N V L R Y K K F G S N V L R Y K K F G I N V L RY K N Y S S N V L R Y K N Y S I N V L R Y K N Y G S N V L R Y K N Y G IN V L R Y K N F S S N V L R Y K N F S I N V L R Y K N F G S N V L R Y KN F G I N V L R Y R K Y S S N V L R Y R K Y S I N V L R Y R K Y G S N VL R Y R K Y G I N V L R Y R K F S S N V L R Y R K F S I N V L R Y R K FG S N V L R Y R K F G I N V L R Y R N Y S S N V L R Y R N Y S I N V L RY R N Y G S N V L R Y R N Y G I N V L R Y R N F S S N V L R Y R N F S IN V L R Y R N F G S N V L R Y R N F G I N V F Y C K K Y S S N V F Y C KK Y S I N V F Y C K K Y G S N V F Y C K K Y G I N V F Y C K K F S S N VF Y C K K F S I N V F Y C K K F G S N V F Y C K K F G I N V F Y C K N YS S N V F Y C K N Y S I N V F Y C K N Y G S N V F Y C K N Y G I N V F YC K N F S S N V F Y C K N F S I N V F Y C K N F G S N V F Y C K N F G IN V F Y C R K Y S S N V F Y C R K Y S I N V F Y C R K Y G S N V F Y C RK Y G I N V F Y C R K F S S N V F Y C R K F S I N V F Y C R K F G S N VF Y C R K F G I N V F Y C R N Y S S N V F Y C R N Y S I N V F Y C R N YG S N V F Y C R N Y G I N V F Y C R N F S S N V F Y C R N F S I N V F YC R N F G S N V F Y C R N F G I N V F Y Y K K Y S S N V F Y Y K K Y S IN V F Y Y K K Y G S N V F Y Y K K Y G I N V F Y Y K K F S S N V F Y Y KK F S I N V F Y Y K K F G S N V F Y Y K K F G I N V F Y Y K N Y S S N VF Y Y K N Y S I N V F Y Y K N Y G S N V F Y Y K N Y G I N V F Y Y K N FS S N V F Y Y K N F S I N V F Y Y K N F G S N V F Y Y K N F G I N V F YY R K Y S S N V F Y Y R K Y S I N V F Y Y R K Y G S N V F Y Y R K Y G IN V F Y Y R K F S S N V F Y Y R K F S I N V F Y Y R K F G S N V F Y Y RK F G I N V F Y Y R N Y S S N V F Y Y R N Y S I N V F Y Y R N Y G S N VF Y Y R N Y G I N V F Y Y R N F S S N V F Y Y R N F S I N V F Y Y R N FG S N V F Y Y R N F G I N V F R C K K Y S S N V F R C K K Y S I N V F RC K K Y G S N V F R C K K Y G I N V F R C K K F S S N V F R C K K F S IN V F R C K K F G S N V F R C K K F G I N V F R C K N Y S S N V F R C KN Y S I N V F R C K N Y G S N V F R C K N Y G I N V F R C K N F S S N VF R C K N F S I N V F R C K N F G S N V F R C K N F G I N V F R C R K YS S N V F R C R K Y S I N V F R C R K Y G S N V F R C R K Y G I N V F RC R K F S S N V F R C R K F S I N V F R C R K F G S N V F R C R K F G IN V F R C R N Y S S N V F R C R N Y S I N V F R C R N Y G S N V F R C RN Y G I N V F R C R N F S S N V F R C R N F S I N V F R C R N F G S N VF R C R N F G I N V F R Y K K Y S S N V F R Y K K Y S I N V F R Y K K YG S N V F R Y K K Y G I N V F R Y K K F S S N V F R Y K K F S I N V F RY K K F G S N V F R Y K K F G I N V F R Y K N Y S S N V F R Y K N Y S IN V F R Y K N Y G S N V F R Y K N Y G I N V F R Y K N F S S N V F R Y KN F S I N V F R Y K N F G S N V F R Y K N F G I N V F R Y R K Y S S N VF R Y R K Y S I N V F R Y R K Y G S N V F R Y R K Y G I N V F R Y R K FS S N V F R Y R K F S I N V F R Y R K F G S N V F R Y R K F G I N V F RY R N Y S S N V F R Y R N Y S I N V F R Y R N Y G S N V F R Y R N Y G IN V F R Y R N F S S N V F R Y R N F S I N V F R Y R N F G S N V F R Y RN F G I N F L Y C K K Y S S N F L Y C K K Y S I N F L Y C K K Y G S N FL Y C K K Y G I N F L Y C K K F S S N F L Y C K K F S I N F L Y C K K FG S N F L Y C K K F G I N F L Y C K N Y S S N F L Y C K N Y S I N F L YC K N Y G S N F L Y C K N Y G I N F L Y C K N F S S N F L Y C K N F S IN F L Y C K N F G S N F L Y C K N F G I N F L Y C R K Y S S N F L Y C RK Y S I N F L Y C R K Y G S N F L Y C R K Y G I N F L Y C R K F S S N FL Y C R K F S I N F L Y C R K F G S N F L Y C R K F G I N F L Y C R N YS S N F L Y C R N Y S I N F L Y C R N Y G S N F L Y C R N Y G I N F L YC R N F S S N F L Y C R N F S I N F L Y C R N F G S N F L Y C R N F G IN F L Y Y K K Y S S N F L Y Y K K Y S I N F L Y Y K K Y G S N F L Y Y KK Y G I N F L Y Y K K F S S N F L Y Y K K F S I N F L Y Y K K F G S N FL Y Y K K F G I N F L Y Y K N Y S S N F L 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N F L R C R N F S S N FL R C R N F S I N F L R C R N F G S N F L R C R N F G I N F L R Y K K YS S N F L R Y K K Y S I N F L R Y K K Y G S N F L R Y K K Y G I N F L RY K K F S S N F L R Y K K F S I N F L R Y K K F G S N F L R Y K K F G IN F L R Y K N Y S S N F L R Y K N Y S I N F L R Y K N Y G S N F L R Y KN Y G I N F L R Y K N F S S N F L R Y K N F S I N F L R Y K N F G S N FL R Y K N F G I N F L R Y R K Y S S N F L R Y R K Y S I N F L R Y R K YG S N F L R Y R K Y G I N F L R Y R K F S S N F L R Y R K F S I N F L RY R K F G S N F L R Y R K F G I N F L R Y R N Y S S N F L R Y R N Y S IN F L R Y R N Y G S N F L R Y R N Y G I N F L R Y R N F S S N F L R Y RN F S I N F L R Y R N F G S N F L R Y R N F G I N F F Y C K K Y S S N FF Y C K K Y S I N F F Y C K K Y G S N F F Y C K K Y G I N F F Y C K K FS S N F F Y C K K F S I N F F Y C K K F G S N F F Y C K K F G I N F F YC K N Y S S N F F Y C K N Y S I N F F Y C K N Y G S N F F Y C K N Y G IN F F Y C K N F S S N F F Y C K N F S I N F F Y C K N F G S N F F Y C KN F G I N F F Y C R K Y S S N F F Y C R K Y S I N F F Y C R K Y G S N FF Y C R K Y G I N F F Y C R K F S S N F F Y C R K F S I N F F Y C R K FG S N F F Y C R K F G I N F F Y C R N Y S S N F F Y C R N Y S I N F F YC R N Y G S N F F Y C R N Y G I N F F Y C R N F S S N F F Y C R N F S IN F F Y C R N F G S N F F Y C R N F G I N F F Y Y K K Y S S N F F Y Y KK Y S I N F F Y Y K K Y G S N F F Y Y K K Y G I N F F Y Y K K F S S N FF Y Y K K F S I N F F Y Y K K F G S N F F Y Y K K F G I N F F Y Y K N YS S N F F Y Y K N Y S I N F F Y Y K N Y G S N F F Y Y K N Y G I N F F YY K N F S S N F F Y Y K N F S I N F F Y Y K N F G S N F F Y Y K N F G IN F F Y Y R K Y S S N F F Y Y R K Y S I N F F Y Y R K Y G S N F F Y Y RK Y G I N F F Y Y R K F S S N F F Y Y R K F S I N F F Y Y R K F G S N FF Y Y R K F G I N F F Y Y R N Y S S N F F Y Y R N Y S I N F F Y Y R N YG S N F F Y Y R N Y G I N F F Y Y R N F S S N F F Y Y R N F S I N F F YY R N F G S N F F Y Y R N F G I N F F R C K K Y S S N F F R 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S T V L Y Y K K F G I T V L Y Y K N Y S S T V L Y Y K N Y S I T V L YY K N Y G S T V L Y Y K N Y G I T V L Y Y K N F S S T V L Y Y K N F S IT V L Y Y K N F G S T V L Y Y K N F G I T V L Y Y R K Y S S T V L Y Y RK Y S I T V L Y Y R K Y G S T V L Y Y R K Y G I T V L Y Y R K F S S T VL Y Y R K F S I T V L Y Y R K F G S T V L Y Y R K F G I T V L Y Y R N YS S T V L Y Y R N Y S I T V L Y Y R N Y G S T V L Y Y R N Y G I T V L YY R N F S S T V L Y Y R N F S I T V L Y Y R N F G S T V L Y Y R N F G IT V L R C K K Y S S T V L R C K K Y S I T V L R C K K Y G S T V L R C KK Y G I T V L R C K K F S S T V L R C K K F S I T V L R C K K F G S T VL R C K K F G I T V L R C K N Y S S T V L R C K N Y S I T V L R C K N YG S T V L R C K N Y G I T V L R C K N F S S T V L R C K N F S I T V L RC K N F G S T V L R C K N F G I T V L R C R K Y S S T V L R C R K Y S IT V L R C R K Y G S T V L R C R K Y G I T V L R C R K F S S T V L R C RK F S I T V L R C R K F G S T V L R C R K F G I T V L R C R N Y S S T VL R C R 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Y C K N F S S T V F Y C K N F S I T V F Y C K N F G S T VF Y C K N F G I T V F Y C R K Y S S T V F Y C R K Y S I T V F Y C R K YG S T V F Y C R K Y G I T V F Y C R K F S S T V F Y C R K F S I T V F YC R K F G S T V F Y C R K F G I T V F Y C R N Y S S T V F Y C R N Y S IT V F Y C R N Y G S T V F Y C R N Y G I T V F Y C R N F S S T V F Y C RN F S I T V F Y C R N F G S T V F Y C R N F G I T V F Y Y K K Y S S T VF Y Y K K Y S I T V F Y Y K K Y G S T V F Y Y K K Y G I T V F Y Y K K FS S T V F Y Y K K F S I T V F Y Y K K F G S T V F Y Y K K F G I T V F YY K N Y S S T V F Y Y K N Y S I T V F Y Y K N Y G S T V F Y Y K N Y G IT V F Y Y K N F S S T V F Y Y K N F S I T V F Y Y K N F G S T V F Y Y KN F G I T V F Y Y R K Y S S T V F Y Y R K Y S I T V F Y Y R K Y G S T VF Y Y R K Y G I T V F Y Y R K F S S T V F Y Y R K F S I T V F Y Y R K FG S T V F Y Y R K F G I T V F Y Y R N Y S S T V F Y Y R N Y S I T V F YY R N Y G S T V F Y Y R N Y G I T V F Y Y R N F S S T V F Y Y R N F S IT V F Y Y R N F G S T V F Y Y R N F G I T V F R C K K Y S S T V F R C KK Y S I T V F R C K K Y G S T V F R C K K Y G I T V F R C K K F S S T VF R C K K F S I T V F R C K K F G S T V F R C K K F G I T V F R C K N YS S T V F R C K N Y S I T V F R C K N Y G S T V F R C K N Y G I T V F RC K N F S S T V F R C K N F S I T V F R C K N F G S T V F R C K N F G IT V F R C R K Y S S T V F R C R K Y S I T V F R C R K Y G S T V F R C RK Y G I T V F R C R K F S S T V F R C R K F S I T V F R C R K F G S T VF R C R K F G I T V F R C R N Y S S T V F R C R N Y S I T V F R C R N YG S T V F R C R N Y G I T V F R C R N F S S T V F R C R N F S I T V F RC R N F G S T V F R C R N F G I T V F R Y K K Y S S T V F R Y K K Y S IT V F R Y K K Y G S T V F R Y K K Y G I T V F R Y K K F S S T V F R Y KK F S I T V F R Y K K F G S T V F R Y K K F G I T V F R Y K N Y S S T VF R Y K N Y S I T V F R Y K N Y G S T V F R Y K N Y G I T V F R Y K N FS S T V F R Y K N F S I T V F R Y K N F G S T V F R Y K N F G I T V F RY R K Y S S T V F R Y R K 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F G I T V L RC R K Y S S T V L R C R K Y S I T V L R C R K Y G S T V L R C R K Y G IT V L R C R K F S S T V L R C R K F S I T V L R C R K F G S T V L R C RK F G I T V L R C R N Y S S T V L R C R N Y S I T V L R C R N Y G S T VL R C R N Y G I T V L R C R N F S S T V L R C R N F S I T V L R C R N FG S T V L R C R N F G I T V L R Y K K Y S S T V L R Y K K Y S I T V L RY K K Y G S T V L R Y K K Y G I T V L R Y K K F S S T V L R Y K K F S IT V L R Y K K F G S T V L R Y K K F G I T V L R Y K N Y S S T V L R Y KN Y S I T V L R Y K N Y G S T V L R Y K N Y G I T V L R Y K N F S S T VL R Y K N F S I T V L R Y K N F G S T V L R Y K N F G I T V L R Y R K YS S T V L R Y R K Y S I T V L R Y R K Y G S T V L R Y R K Y G I T V L RY R K F S S T V L R Y R K F S I T V L R Y R K F G S T V L R Y R K F G IT V L R Y R N Y S S T V L R Y R N Y S I T V L R Y R N Y G S T V L R Y RN Y G I T V L R Y R N F S S T V L R Y R N F S I T V L R Y R N F G S T VL R Y R N F G I T V F Y C K K Y S S T V F Y C K K Y S I T V F Y C K K YG S T V F Y C K K Y G I T V F Y C K K F S S T V F Y C K K F S I T V F YC K K F G S T V F Y C K K F G I T V F Y C K N Y S S T V F Y C K N Y S IT V F Y C K N Y G S T V F Y C K N Y G I T V F Y C K N F S S T V F Y C KN F S I T V F Y C K N F G S T V F Y C K N F G I T V F Y C R K Y S S T VF Y C R K Y S I T V F Y C R K Y G S T V F Y C R K Y G I T V F Y C R K FS S T V F Y C R K F S I T V F Y C R K F G S T V F Y C R K F G I T V F YC R N Y S S T V F Y C R N Y S I T V F Y C R N Y G S T V F Y C R N Y G IT V F Y C R N F S S T V F Y C R N F S I T V F Y C R N F G S T V F Y C RN F G I T V F Y Y K K Y S S T V F Y Y K K Y S I T V F Y Y K K Y G S T VF Y Y K K Y G I T V F Y Y K K F S S T V F Y Y K K F S I T V F Y Y K K FG S T V F Y Y K K F G I T V F Y Y K N Y S S T V F Y Y K N Y S I T V F YY K N Y G S T V F Y Y K N Y G I T V F Y Y K N F S S T V F Y Y K N F S IT V F Y Y K N F G S T V F Y Y K N F G I T V F Y Y R K Y S S T V F Y Y RK Y S I T V F Y Y R K Y G S T V F Y Y R K Y G I T V F Y Y R K F S S T VF Y Y R K F S I T V F Y Y R K F G S T V F Y Y R K F G I T V F Y Y R N YS S T V F Y Y R N Y S I T V F Y Y R N Y G S T V F Y Y R N Y G I T V F YY R N F S S T V F Y Y R N F S I T V F Y Y R N F G S T V F Y Y R N F G IT V F R C K K Y S S T V F R C K K Y S I T V F R C K K Y G S T V F R C KK Y G I T V F R C K K F S S T V F R C K K F S I T V F R C K K F G S T VF R C K K F G I T V F R C K N Y S S T V F R C K N Y S I T V F R C K N YG S T V F R C K N Y G I T V F R C K N F S S T V F R C K N F S I T V F RC K N F G S T V F R C K N F G I T V F R C R K Y S S T V F R C R K Y S IT V F R C R K Y G S T V F R C R K Y G I T V F R C R K F S S T V F R C RK F S I T V F R C R K F G S T V F R C R K F G I T V F R C R N Y S S T VF R C R N Y S I T V F R C R N Y G S T V F R C R N Y G I T V F R C R N FS S T V F R C R N F S I T V F R C R N F G S T V F R C R N F G I T V F RY K K Y S S T V F R Y K K Y S I T V F R Y K K Y G S T V F R Y K K Y G IT V F R Y K K F S S T V F R Y K K F S I T V F R Y K K F G S T V F R Y KK F G I T V F R Y K N Y S S T V F R Y K N Y S I T V F R Y K N Y G S T VF R Y K N Y G I T V F R Y K N F S S T V F R Y K N F S I T V F R Y K N FG S T V F R Y K N F G I T V F R Y R K Y S S T V F R Y R K Y S I T V F RY R K Y G S T V F R Y R K Y G I T V F R Y R K F S S T V F R Y R K F S IT V F R Y R K F G S T V F R Y R K F G I T V F R Y R N Y S S T V F R Y RN Y S I T V F R Y R N Y G S T V F R Y R N Y G I T V F R Y R N F S S T VF R Y R N F S I T V F R Y R N F G S T V F R Y R N F G I T F L Y C K K YS S T F L Y C K K Y S I T F L Y C K K Y G S T F L Y C K K Y G I T F L YC K K F S S T F L Y C K K F S I T F L Y C K K F G S T F L Y C K K F G IT F L Y C K N Y S S T F L Y C K N Y S I T F L Y C K N Y G S T F L Y C KN Y G I T F L Y C K N F S S T F L Y C K N F S I T F L Y C K N F G S T FL Y C K N F G I T F L Y C R K Y S S T F L Y C R K Y S I T F L Y C R K YG S T F L Y C R K Y G I T F L Y C R K F S S T F L Y C R K F S I T F L YC R K F G S T F L Y C R K F G I T F L Y C R N Y S S T F L Y C R N Y S IT F L Y C R N Y G S T F L Y C R N Y G I T F L Y C R N F S S T F L Y C RN F S I T F L Y C R N F G S T F L Y C R N F G I T F L Y Y K K Y S S T FL Y Y K K Y S I T F L Y Y K K Y G S T F L Y Y K K Y G I T F L Y Y K K FS S T F L Y Y K K F S I T F L Y Y K K F G S T F L Y Y K K F G I T F L YY K N Y S S T F L Y Y K N Y S I T F L Y Y K N Y G S T F L Y Y K N Y G IT F L Y Y K N F S S T F L Y Y K N F S I T F L Y Y K N F G S T F L Y Y KN F G I T F L Y Y R K Y S S T F L Y Y R K Y S I T F L Y Y R K Y G S T FL Y Y R K Y G I T F L Y Y R K F S S T F L Y Y R K F S I T F L Y Y R K FG S T F L Y Y R K F G I T F L Y Y R N Y S S T F L Y Y R N Y S I T F L YY R N Y G S T F L Y Y R N Y G I T F L Y Y R N F S S T F L Y Y R N F S IT F L Y Y R N F G S T F L Y Y R N F G I T F L R C K K Y S S T F L R C KK Y S I T F L R C K K Y G S T F L R C K K Y G I T F L R C K K F S S T FL R C K K F S I T F L R C K K F G S T F L R C K K F G I T F L R C K N YS S T F L R C K N Y S I T F L R C K N Y G S T F L R C K N Y G I T F L RC K N F S S T F L R C K N F S I T F L R C K N F G S T F L R C K N F G IT F L R C R K Y S S T F L R C R K Y S I T F L R C R K Y G S T F L R C RK Y G I T F L R C R K F S S T F L R C R K F S I T F L R C R K F G S T FL R C R K F G I T F L R C R N Y S S T F L R C R N Y S I T F L R C R N YG S T F L R C R N Y G I T F L R C R N F S S T F L R C R N F S I T F L RC R N F G S T F L R C R N F G I T F L R Y K K Y S S T F L R Y K K Y S IT F L R Y K K Y G S T F L R Y K K Y G I T F L R Y K K F S S T F L R Y KK F S I T F L R Y K K F G S T F L R Y K K F G I T F L R Y K N Y S S T FL R Y K N Y S I T F L R Y K N Y G S T F L R Y K N Y G I T F L R Y K N FS S T F L R Y K N F S I T F L R Y K N F G S T F L R Y K N F G I T F L RY R K Y S S T F L R Y R K Y S I T F L R Y R K Y G S T F L R Y R K Y G IT F L R Y R K F S S T F L R Y R K F S I T F L R Y R K F G S T F L R Y RK F G I T F L R Y R N Y S S T F L R Y R N Y S I T F L R Y R N Y G S T FL R Y R N Y G I T F L R Y R N F S S T F L R Y R N F S I T F L R Y R N FG S T F L R Y R N F G I T F F Y C K K Y S S T F F Y C K K Y S I T F F YC K K Y G S T F F Y C K K Y G I T F F Y C K K F S S T F F Y C K K F S IT F F Y C K K F G S T F F Y C K K F G I T F F Y C K N Y S S T F F Y C KN Y S I T F F Y C K N Y G S T F F Y C K N Y G I T F F Y C K N F S S T FF Y C K N F S I T F F Y C K N F G S T F F Y C K N F G I T F F Y C R K YS S T F F Y C R K Y S I T F F Y C R K Y G S T F F Y C R K Y G I T F F YC R K F S S T F F Y C R K F S I T F F Y C R K F G S T F F Y C R K F G IT F F Y C R N Y S S T F F Y C R N Y S I T F F Y C R N Y G S T F F Y C RN Y G I T F F Y C R N F S S T F F Y C R N F S I T F F Y C R N F G S T FF Y C R N F G I T F F Y Y K K Y S S T F F Y Y K K Y S I T F F Y Y K K YG S T F F Y Y K K Y G I T F F Y Y K K F S S T F F Y Y K K F S I T F F YY K K F G S T F F Y Y K K F G I T F F Y Y K N Y S S T F F Y Y K N Y S IT F F Y Y K N Y G S T F F Y Y K N Y G I T F F Y Y K N F S S T F F Y Y KN F S I T F F Y Y K N F G S T F F Y Y K N F G I T F F Y Y R K Y S S T FF Y Y R K Y S I T F F Y Y R K Y G S T F F Y Y R K Y G I T F F Y Y R K FS S T F F Y Y R K F S I T F F Y Y R K F G S T F F Y Y R K F G I T F F YY R N Y S S T F F Y Y R N Y S I T F F Y Y R N Y G S T F F Y Y R N Y G IT F F Y Y R N F S S T F F Y Y R N F S I T F F Y Y R N F G S T F F Y Y RN F G I T F F R C K K Y S S T F F R C K K Y S I T F F R C K K Y G S T FF R C K K Y G I T F F R C K K F S S T F F R C K K F S I T F F R C K K FG S T F F R C K K F G I T F F R C K N Y S S T F F R C K N Y S I T F F RC K N Y G S T F F R C K N Y G I T F F R C K N F S S T F F R C K N F S IT F F R C K N F G S T F F R C K N F G I T F F R C R K Y S S T F F R C RK Y S I T F F R C R K Y G S T F F R C R K Y G I T F F R C R K F S S T FF R C R K F S I T F F R C R K F G S T F F R C R K F G I T F F R C R N YS S T F F R C R N Y S I T F F R C R N Y G S T F F R C R N Y G I T F F RC R N F S S T F F R C R N F S I T F F R C R N F G S T F F R C R N F G IT F F R Y K K Y S S T F F R Y K K Y S I T F F R Y K K Y G S T F F R Y KK Y G I T F F R Y K K F S S T F F R Y K K F S I T F F R Y K K F G S T FF R Y K K F G I T F F R Y K N Y S S T F F R Y K N Y S I T F F R Y K N YG S T F F R Y K N Y G I T F F R Y K N F S S T F F R Y K N F S I T F F RY K N F G S T F F R Y K N F G I T F F R Y R K Y S S T F F R Y R K Y S IT F F R Y R K Y G S T F F R Y R K Y G I T F F R Y R K F S S T F F R Y RK F S I T F F R Y R K F G S T F F R Y R K F G I T F F R Y R N Y S S T FF R Y R N Y S I T F F R Y R N Y G S T F F R Y R N Y G I T F F R Y R N FS S T F F R Y R N F S I T F F R Y R N F G S T F F R Y R N F G I

Example 9 Inhibition of Heregulin-β Induced ErbB2 Phosphorylation andCell Proliferation in ErbB2 Low Expressing MCF7 Cells

As described in Example 4, the hybridoma supernatants could inhibitHeregulin-induced ErbB2 phosphorylation in MCF7 cells. Using purifiedmonoclonal antibodies, the potency of the ErbB2 antibodies wasdetermined, also compared to two inhibitory antibodies 2C4 andHerceptin®. Briefly, MCF7 cells were seeded at 20,000 cells/well in 96well plates and cultured in full growth media (10% FCS) overnight. Thenext day, cell culture plates were washed once with PBS and culturemedium was replaced with phenol red-free and serum-free medium. Cellswere serum-starved overnight, and then were incubated with mAbs,Herceptin®, or 2C4 titrating 1:5 from 10 μg/ml in serum-free medium for1 hour before being treated with 10 nM Heregulin-β for 10 minutes. Celllysates were prepared as described in Example 4 and Phosphor-ErbB2 levelwas measured using an ELISA kit from RnD systems (human Phospho-ErbB2DuoSet IC, Cat #DYC1768), according to the protocol provided. Percentageof inhibition was calculated based on the level of pErbB2 inun-stimulated cells and Heregulin-stimulated cells in the absence ofantibodies. A dose response curve was plotted for each antibody usingthe PrismGraphpad software.

FIG. 3 illustrates the dose response curves from a representativeexperiment. EC50 values were derived from nonlinear regression analysis,and shown in Table 7. 2C4 inhibited ErbB2 phosphorylation whileHerceptin® had little effect. Eight out of 11 monoclonal antibodies ofthe invention tested showed inhibitory activity on ErbB2 phosphoryaltionin MCF7 cells. 1.18.1 appears to have better potency than 2C4.

TABLE 7 Potency of 10 purified mAbs of the invention at inhibitingHeregulin-induced ErbB2 phophorylation in MCF7 cells. mAbs EC50 (ng/ml)n1 EC50 (ng/ml) n2 2C4 43.6 14.6 Herceptin ® >10,000 >10,000 1.18.1 4.210.8 1.20.1 15.5 17.4 1.140.1 14.65 22.9 1.96.2 28.8 34 1.100.1 39.933.7 1.14.1 33.7 22.9 1.39.1 46.9 26.3 1.24.3 42.9 20.41.43.1 >10,000 >10,000 1.44.1 >10,000 >10,000 1.71.3 >10,000 nd

The effect of the monoclonal antibodies on Heregulin-induced cellproliferation was also examined. MCF7 cells were seeded at 6000cell/well in 96 well plates in Phenol red free DMEM media with 10% FCS,NaPyruvate, L-Glutamine, and allowed to grow overnight at 37° C. Thenext day, cells were washed once with cold PBS and culture media wasreplaced with 100 μl of FCS/phenol red free media plus NaPyruvate andincubated for 4 hours at 37° C. Then, FCS-free media was removed and 50μl of titrated mAbs and 50 μl of 2 nM Heregulin-β were added to cells.After incubating cells with antibodies and Heregulin for three days at37° C., 25 μl of CellTiter-Glo luminescent reagent (Promega, catalog#07570) was added to each well. Plates were agitated for 5 minutes andthen incubated for 10 minutes at room temperature. Luminescence was readout on a microtiterplate luminometer (Tecan GENios Pro). Percentage ofinhibition was calculated based on the level of luminescence inun-stimulated cells and Heregulin-stimulated cells in the absence ofantibodies.

As shown in FIG. 4, a dose response curve was plotted for each antibodyusing the PrismGraphpad software. EC50 values were derived fromnonlinear regression analysis, and listed in Table 8. As exhibited inthe ErbB2 phosphorylation assay, 2C4 and the same 8 antibodies of theinvention that were effective at inhibiting ErbB2 phosphorylation showeddose-dependent inhibition of Heregulin-induced MCF7 cell proliferationwhile Herceptin® and the 3 antibodies ineffective at blocking ErbB2phosphorylation did not.

TABLE 8 Potency and efficacy of 10 purified mAbs of the invention atinhibiting Heregulin-induced MCF7 cell proliferation mAbs EC50 (ng/ml) %Inhibition @10 μg/ml 2C4 404.5 90 Herceptin ® >10,000 39 1.18.1 414.8 941.20.1 770.1 100 1.140.1 633.3 91 1.96.2 2832 98 1.100.1 2043 95 1.14.1640 96 1.39.1 479.3 89 1.24.3 3017 81 1.43.1 ~10,000 53 1.44.1 >10,00018 1.71.3 >10,000 nd

Example 10 Inhibition of Proliferation of BT474 and SKBR3Cells

The 8 neutralizing antibodies effective on ErbB2-low expressing cells(MCF7) were also examined for their ability to inhibit ErbB2-highexpressing cells in a 4-day cell proliferation assay. Five thousandcells (BT474 or SKBR3) in 50 μl of growth media with 10% FCS were seededin 96 well plates and incubated at 37° C. for 4 hours in order to attachto plates. Monoclonal antibodies were titrated 1:5 in growth media at 2×final concentrations starting from 40 μg/ml. Fifty (50) of 2C4,Herceptin® and 8 anti-ErbB2 mAbs of the invention were added to theplates and cells were cultured with antibodies for 4 days. Twenty-five(25) μl of CellTiter-glo reagent was added to each well. Plates wereagitated for 5 minutes and incubated for 10 minutes at room temperature.Luminescence was read out on a microtiterplate luminometer (Teem GENiosPro). Percentage of inhibition was calculated based on the level ofluminescence signal on day 4 and day 0 in the absence of antibodies. Adose response curve was plotted for each antibody using thePrismGraphpad software, and EC50 values were derived from nonlinearregression analysis.

FIGS. 5 and 6 illustrate data from a representative experiment. BothEC50 and maximum percentage of inhibition are listed in Table 9 and 10.As reported, Herceptin® showed dose-dependent inhibition of both BT474and SKBR3 cell proliferation while 2C4 had little effect. All of themonoclonal antibodies of the invention tested exhibited dose-dependentinhibition of BT474 and SKBR3 cell proliferation with comparable potencyto Herceptin®.

TABLE 9 Potency and efficacy of 8 antibodies of the invention atinhibiting BT474 cell proliferation N1 EC50 N1% Inhibition N2 EC50 N2%Inhibition Abs (ng/ml) @40 μg/ml (ng/ml) @40 μg/ml Herceptin ® 144.8 6181.8 57 2C4 >40,000 −9 4750.0 18 hIgG1 >40,000 −2 >40,000 10 1.96.2131.3 35 66.3 29 1.20.1 85.3 40 12.8 37 1.140.1 299.8 39 70.9 45 1.18.138.1 34 28.2 39 1.100.1 169.4 35 91.1 32 1.24.3 198.3 24 74.4 29 1.14.1135.7 38 63.0 39 1.39.1 156.4 29 42.3 40

TABLE 10 Potency and efficacy of 8 antibodies of the invention atinhibiting SKBR3 cell proliferation Abs EC50 (ng/ml) % inhibition @36μg/ml Herceptin ® 54.5 59 2C4 >36,000 18 hIgG1 >36,000 2 1.96.2 41.7 431.20.1 25.2 49 1.140.1 42.7 44 1.18.1 26.3 46 1.100.1 61.3 54 1.24.348.5 37 1.14.1 35.7 52 1.39.1 68.9 41

Example 11 Inhibition of ErbB2 Phosphorylation in BT474 Cells

To identify the mechanism of action of these antibodies in ErbB2-highexpressing cell lines, an antibody of the invention 1.18.1, was testedfor effect on constitutive ErbB2 phosphorylation in BT474 cells alongwith Herceptin® and 2C4. BT474 cells were seeded into 96 well cultureplates in complete culture media at 5000 cells/well on day 1, 10000cells/well on day 2 and day 3, or at 20000 cells/well on day 4. Cellswere incubated at 37° C. for 3-4 hours and allowed to attach to plates.Monoclonal antibodies were titrated 1:5 in complete media starting from10 μg/ml for 6 points and then added to cells. Cells were incubated withmonoclonal antibodies for 1-4 days. On day 5, cells were lysed in buffersupplemented with protease and phosphatase inhibitors as previouslydescribed. Phospho-ErbB2 levels in cell lysates were determined byELISA. Cell proliferation was measured by CyQuant (Invitrogen).Percentage of inhibition was calculated according to the pErB2 level inthe absence of antibodies. Phosphor-ErbB2/cell was also calculated bynormalizing phosphor-ErbB2 levels to cell numbers. A dose response curvewas plotted for each antibody using the PrismGraphpad software, and EC50values were derived from nonlinear regression analysis.

FIGS. 7 and 8 illustrate the dose response at different time points.Both EC50 and maximum percentage of inhibition were listed in Table 11.Monoclonal Ab 1.18.1 started to show inhibition of pErB2 at 48 hrs andreached the maximal inhibition at 72 hours. However, Herceptin® did notshow a significant effect until 72 hrs while 2C4 had little effect.Interestingly, when phosphor-ErbB2 levels were normalized by cellnumber, inhibition of ErbB2 phosphorylation by 1.18.1 reached maximum at48 hours; while Herceptin® did not appear to inhibit phosphorylation ofErbB2, as previously published.

Next, the effect of 7 of the antibodies on constitutive ErbB2phosphorylation in BT474 cells after 48 hours was investigated. Briefly,10,000 BT474 cells in 50 μl of complete culture media was seeded in 96well plates and cultured for 3-4 hours at 37° C. to attach to theplates. Then 50 μl of 20 ug/ml 2C4, Herceptin® and anti-ErbB2 monoclonalantibodies of the invention, prepared at 2× final concentrations incomplete culture media, was added to the plates and incubated with cellsfor 48 hours. Cell lysates were prepared and phosphor-ErbB2 levels weremeasured by ELISA as described in Example 4. Percentage of inhibitionwas calculated according to the pErB2 level in the absence ofantibodies. As listed in Table 11, all 7 mAbs of the invention exhibiteddose-dependent inhibition of ErbB2 phosphorylation in BT474 cells at 48hours. In comparison, Herceptin®, and 2C4 had little effect.

TABLE 11 Potency of 7 antibodies of the invention at inhibiting ErbB2phosphorylation in BT474 cell at 48 hours % Inhibition @10 % Inhibition@10 Abs μg/ml (n1) μg/ml (n2) Herceptin ® 5 10 2C4 10 7 1.96.2 37 301.20.1 21 28 1.140.1 31 33 1.18.1 24 33 1.100.1 37 26 1.14.1 37 451.39.1 37 26

Example 12 Determination of Anti-ErbB2 Antibody Affinity Using (A)Medium Resolution Biacore Analysis

The binding affinity of eight of the anti-ErbB2 antibodies was measuredby medium resolution Biacore. All experiments were performed using aBiacore 2000 instrument.

First, 12 high-density goat α-human IgG antibody surfaces over three CM5Biacore chips were prepared using routine amine coupling. Then, mAbswere diluted in HBS-P running buffer containing 100 μg/ml Bovine serumalbumin (BSA), specifically mAb 1.18.1 to 11 μg/mL, mAb 1.20.1 to 9.9μg/mL, mAb 1.100.1 to 11 μg/mL, mAb 1.96.2 to 9.3 μg/mL, mAb 1.140.1 to9.2 μg/mL, mAb 1.14.1 to 9.3 μg/mL, mAb 1.39.1 to 10 μg/mL, and mAb1.24.3 to 10 μg/mL. Before each antigen injection cycle, each mAb wascaptured for six to nine seconds at a 100 μL/min flow rate. A 2-minutewash step followed each capture injection to stabilize each mAbbaseline. Purified human ErbB2(ECD)-cMyc/His was injected for 90 secondsat a concentration range of 307-4.80 nM (2× serial dilution) for allmAbs, followed by a 15 minute dissociation except for mAbs 1.39.1 and1.24.3 where dissociation was followed for 20 mins. All samples wererandomly injected with several mAb capture/buffer inject cyclesinterspersed for double referencing. The high-density goat α-humanantibody surfaces were regenerated with one 12-second pulse of 146 mMphosphoric acid (pH 1.5) after each cycle. A flow rate of 100 μL/min.was used for all injection cycles. The data was fit to a 1:1 interactionmodel using CLAMP. The resulting binding constants are listed in thetable below. MAbs are listed in order from highest to lowest affinity.

TABLE 12(a) Binding affinities of 8 antibodies of the invention againsthuman ErbB2 by Medium resolution Biacore analysis mAbs R_(max) k_(a)(M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (nM) 1.39.1 57 1.15 × 10⁵ 2.36 × 10⁻⁴ 2.01.14.1 70 1.02 × 10⁵ 2.56 × 10⁻⁴ 2.5 1.96.2 51 1.03 × 10⁵ 2.73 × 10⁻⁴2.6 1.100.1 100 1.04 × 10⁵ 3.01 × 10⁻⁴ 2.9 1.18.1 157 1.00 × 10⁵ 3.02 ×10⁻⁴ 3.0 1.140.1 66 0.94 × 10⁵ 3.33 × 10⁻⁴ 3.5 1.20.1 56 0.95 × 10⁵ 3.44× 10⁻⁴ 3.6 1.24.3 65 1.28 × 10⁵ 6.55 × 10⁻⁴ 5.1

(B) High Resolution Biacore Analysis

All experiments were performed using a Biacore T100 instrument. First, ahigh-density goat α human IgG antibody (Caltag H10500) surface wasprepared over two CM5 Biacore chips using routine amine coupling. EachmAb was diluted in HBS-P running buffer containing 100 μg/ml Bovineserum albumin (BSA). MAb 1.140 was diluted to 5 μg/mL, mAb 1.96.2 to 5.9μg/mL, mAb 1.39.1 to 8.7 μg/mL, mAb 2C4 to 2 μg/mL, and herceptin to 4μg/mL.

A capture level protocol was developed for all five mAbs. Before eachantigen injection cycle, each mAb was captured for 15 to 30 seconds at a20 μL/min flow rate. A 5-minute wash step followed each captureinjection to stabilize each mAb baseline. Antigen hHer-2(ECD)cMyc (Lot#452) was injected for 4 minutes at a concentration range of 369-5.76 nM(2× serial dilution) for mAbs 1.140, 1.96.2, and 1.39.1 followed by a 15minute dissociation, and a concentration range of 650-10.2 nM (2× serialdilution) for mAb 2C4 and herceptin followed by a 25 minutedissociation. The samples were prepared in the running buffer describedabove. All samples were randomly injected with several mAbcapture/buffer inject cycles interspersed for double referencing. Thehigh-density goat α-human antibody surfaces were regenerated with one15-second pulse of 146 mM phosphoric acid (pH 1.5) after each cycle. Aflow rate of 50 μL/min. was used for all injection cycles.

All sensorgram data were fit to a 1:1 interaction model using CLAMP. Theresulting binding constants are listed in the table below. MAbs arelisted in order from highest to lowest affinity.

TABLE 12(b) Binding affinities of 3 antibodies of the invention againsthuman ErbB2 by high resolution Biacore analysis Sample R_(max) k_(a)(M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (nM) 2C4 105, 88 1.21 × 10⁴ 3.91 × 10⁻⁵ 3.2herceptin 60 2.34 × 10⁴ 9.50 × 10⁻⁵ 4.2 1.140 110 1.64 × 10⁴ 1.73 × 10⁻⁴10.6 1.96.2 93 1.68 × 10⁴ 1.94 × 10⁻⁴ 11.6 1.39.1 120 1.60 × 10⁴ 2.14 ×10⁻⁴ 13.4

Example 13 Competition Binning of Antibodies

2C4 reportedly binds to the dimerization domain on ErbB2 whileHerceptin® binds to the C-terminal domain in the extracellular region ofErbB2 (see Franklin et al., Cancer Cell. 2004 April; 5(4):317-28 and Choet al., Nature. 2003 Feb. 13; 421(6924):756-60, hereby incorporated byreference). To determine if the binding epitopes of 8 of the antibodiesoverlap with the epitopes for 2C4 or Herceptin®, competitive binningELISA was performed.

Costar 3695 medium binding 96 well plates were coated with 0.5 μg/ml.Herceptin® or 2 μg/ml 2C4 in PBS overnight at 4° C. Coated plates werewashed and then blocked with 1% milk/PBS for 30 minutes at roomtemperature (RT). Antibodies were titrated to 1000 ng/ml, 100 ng/ml, and10 ng/ml and pre-incubated with 30 ng/ml hErbB2 (ECD) for 2 hours. Theantibody/ErbB2 mixture was transferred to blocked plates and incubatedfor 1 hour at RT. To detect bound ErbB2, the plates were and washed andincubated with 1 μg/ml goat anti-ErbB2 (R&D systems, catalog #AF1129)for 1 hour at RT. Secondary antibody rabbit anti-goat IgG Fc POD (Piercecatalog #31433, 400 ng/ml) was added to the plates and incubated for 1hour at RT. After extensive washing, substrate TMB (Neogen) was addedand incubated with the plates for 10-18 minutes at RT. The enzymereaction was stopped by the addition of 1N HCl, and optical density at450 nm was read on a microplate reader. FIG. 9 illustrates the bindingability of ErbB2 to Herceptin® (A) and 2C4 (B) in the presence of 8anti-ErbB2 antibodies of the invention, mAb 1.71.3, or an irrelevantisotope control mAb generated in-house. None of the anti-ErbB2antibodies of the invention block ErbB2 binding to 2C4 or Herceptin®,suggesting that the antibodies belong to a different bin than 2C4 orHerceptin®.

All publications, patents and patent applications herein areincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. The foregoing detaileddescription has been given for clearness of understanding only and nounnecessary limitations should be understood therefrom as modificationswill be obvious to those skilled in the art. It is not an admission thatany of the information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A monoclonal antibody or an antigen-bindingfragment thereof that specifically binds ErbB2 wherein the antibody orantigen binding fragment thereof comprises: (a) heavy chain CDR1, CDR2and CDR3 amino acid sequences of SEQ ID NO: 34; and (b) light chainCDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 36, wherein thereare one to three amino acid substitutions among said CDRs selected fromthe group consisting of: (i) substitution of a cysteine residue with anon-cysteine amino acid residue; (ii) substitution of a methionine toeliminate a potential oxidation site; and (iii) substitution of a lightchain non-germline residue with a corresponding light chain germlineresidue.
 2. The monoclonal antibody or an antigen-binding fragmentthereof of claim 1, wherein the heavy chain CDR1 comprises thesubstitution of a methionine to eliminate a potential oxidation site. 3.The, monoclonal antibody or an antigen-binding fragment thereof of claim1, wherein the light chain CDR1 comprises the substitution of a cysteineresidue with a non-Cysteine amino acid residue.
 4. The monoclonalantibody or antigen-binding fragment thereof of claim 1, wherein theantibody or antigen-binding fragment thereof is selected from the groupconsisting of a chimeric antibody, human antibody and humanizedantibody.
 5. The monoclonal antibody or antigen-binding fragment thereofof claim 2, wherein the antibody or antigen-binding fragment thereof isselected from the group consisting of a chimeric antibody, humanantibody and humanized antibody.
 6. The monoclonal antibody orantigen-binding fragment thereof of claim 3, wherein the antibody orantigen-binding fragment thereof is selected from the group consistingof a chimeric antibody, human antibody and humanized antibody.
 7. Acomposition comprising the monoclonal antibody or antigen-bindingfragment thereof of claim 1 and a pharmaceutically acceptable carrier.8. A composition comprising the monoclonal antibody or antigen-bindingfragment thereof of claim 2 and a pharmaceutically acceptable carrier.9. A composition comprising the monoclonal antibody or antigen-bindingfragment thereof of claim 3 and a pharmaceutically acceptable carrier.10. A composition comprising the monoclonal antibody or antigen-bindingfragment thereof of claim 4 and a pharmaceutically acceptable carrier.11. A composition comprising the monoclonal antibody or antigen-bindingfragment thereof of claim 5 and a pharmaceutically acceptable carrier.12. A composition comprising the monoclonal antibody or antigen-bindingfragment thereof of claim 6 and a pharmaceutically acceptable carrier.